ALBERT

Beschreibung: Key Points AML1-ETO-W692A loses interaction between NHR4 and N-CoR, decreases AML1-ETO cellular dysregulation, and promotes leukemia development in mice.

Beschreibung: Advancements in human pluripotent stem cell (hPSC) research have potential to revolutionize therapeutic transplantation. It has been demonstrated that transcription factors may play key roles in regulating maintenance, expansion, and differentiation of hPSCs. In addition to its regulatory functions in hematopoiesis and blood-related disorders, the transcription factor RUNX1 is also required for the formation of definitive blood stem cells. In this study, we demonstrated that expression of endogenous RUNX1a, an isoform of RUNX1, parallels with lineage commitment and hematopoietic emergence from hPSCs, including both human embryonic stem cells and inducible pluripotent stem cells. In a defined hematopoietic differentiation system, ectopic expression of RUNX1a facilitates emergence of hematopoietic progenitor cells (HPCs) and positively regulates expression of mesoderm and hematopoietic differentiation-related factors, including Brachyury, KDR, SCL, GATA2, and PU.1. HPCs derived from RUNX1a hPSCs show enhanced expansion ability, and the ex vivo–expanded cells are capable of differentiating into multiple lineages. Expression of RUNX1a in embryoid bodies (EBs) promotes definitive hematopoiesis that generates erythrocytes with β-globin production. Moreover, HPCs generated from RUNX1a EBs possess ≥9-week repopulation ability and show multilineage hematopoietic reconstitution in vivo. Together, our results suggest that RUNX1a facilitates the process of producing therapeutic HPCs from hPSCs.

Beschreibung: RUNX1-ETO (also known as AML1-ETO and AML1-MTG8) is a fusion gene generated from t(8;21), which is a common chromosome translocation in acute myeloid leukemia (AML). It has been shown that t(8;21) requires additional aberrations to induce leukemia. Interestingly, 32-59% of t(8;21) patients also display loss of a sex chromosome (LOS) in their leukemia cells. Therefore, loss of the genes located on the sex chromosomes, especially in the pseudoautosomal regions (PARs) that are shared between the X and Y chromosomes, may contribute to RUNX1-ETO leukemia development. One gene of interest in the PARs is CSF2RA, which encodes the alpha subunit of the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor. When the GM-CSF receptor is bound to its ligand, downstream signaling events promote various functional outcomes including proliferation, differentiation, self-renewal, and survival of myeloid cells. Thus, GM-CSF signaling has the potential to regulate both normal and malignant hematopoiesis. We previously reported that mice expressing RUNX1-ETO in GM-CSF deficient hematopoietic cells displayed higher incidence of leukemia (Matsuura S et al. 2012 Blood 119:3155). This result suggests that GM-CSF signaling is inhibitory to RUNX1-ETO dependent leukemogenesis. Furthermore, GM-CSF treatment reduces the self-renewal potential of RUNX1-ETO expressing cells and promotes myeloid differentiation in replating assays. We therefore hypothesize that the negative effect of GM-CSF on RUNX1-ETO induced leukemia development is due to the activation of selected GM-CSF downstream signaling pathway(s) that diminish self-renewal capacity and promote myeloid differentiation. To understand the molecular mechanism of the negative effect of GM-CSF on t(8;21) leukemogenesis, in the current report, we conducted a gene expression profiling assay to examine the effect of GM-CSF on RUNX1-ETO cells. MigR1 vector control or MigR1-RUNX1-ETO retrovirus transduced lineage negative/c-Kit positive (Lin-/c-Kit+) murine hematopoietic stem/progenitor cells (HSPCs) were cultured with or without GM-CSF for 24 hours. Then, Lin-/c-Kit+/GFP+ HSPCs were isolated for the profiling study. We observed little response to GM-CSF in control HSPCs, with only 4 genes being differentially expressed after a 2-fold cutoff. Conversely, 122 genes were differentially expressed in RUNX1-ETO cells treated with GM-CSF. These results clearly indicate that RUNX1-ETO specifically enhances GM-CSF responsiveness in HSPCs. Gene Set Enrichment Analysis (GSEA) of the differentially expressed genes in RUNX1-ETO cells reveals that this response resembles that of GM-CSF-induced myeloid differentiation. Furthermore, pathway analysis of these differentially expressed genes predicts MEK1/2 and ERK1/2 to be activated after GM-CSF treatment in RE cells. We previously reported that ERK1/2, downstream targets of MEK1/2, are hyper-phosphorylated after GM-CSF treatment of RUNX1-ETO cells, and MEK-ERK activation has been shown to regulate cell proliferation and myelopoiesis. Other GM-CSF induced genes are predicted targets of MYD88. MYD88 is upregulated during myeloid differentiation. Its in vivo knockout has been reported to result in an increase of hematopoietic stem cells (HSCs) and reduction of mature granulocytes. Most interestingly, a subset of genes upregulated in GM-CSF treated RUNX1-ETO cells are predicted to be activated by CEBPβ. CEBPβ can heterodimerize with CEBPα and is induced during myelopoiesis, critical for macrophage differentiation, capable of promoting granulopoiesis, and involved in regulating granulopoiesis in vivo. In conclusion, our data suggest that RUNX1-ETO expression results in hyper-responsiveness to GM-CSF. Such enhanced GM-CSF signaling activates the expression of a specific group of genes and results in the reduced self-renewal capacity and increased myeloid differentiation of HSPCs. These GM-CSF effects are likely involved in reducing the leukemogenic potential of RUNX1-ETO and may be considered for specific therapeutic interventions. Disclosures: No relevant conflicts of interest to declare.

Beschreibung: 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.

Beschreibung: Introduction The t(8;21) chromosomal translocation is one of the most common chromosomal translocations associated with acute myeloid leukemia (AML), present in greater than 10% of de novo AML cases. Most of these t(8;21) AML cases are classified as FAB subtype M2. This translocation results in the formation of a stable fusion protein made up of portions of the RUNX1 (aka AML1) and ETO (aka MTG8 and RUNX1T1) proteins. RUNX1 is a transcription factor that is essential for regulating the differentiation of hematopoietic cells, and the fusion protein retains its DNA-binding domain. Additionally, ETO contains four Nervy homology (NH) domains which facilitate a number of protein-protein interactions, notably with the NCOR2/SMRT co-repressor complex. The identification of individual genes or biological pathways which are specifically disrupted in the presence of RUNX1-ETO will provide further molecular insight into the pathogenesis of t(8;21)+ AML and lead to the possibility for improved treatment for these patients. Methods/Results We analyzed publicly available gene expression microarray datasets (Oncomine, TCGA) to search for genes whose expression was significantly altered in the blood of t(8;21)+ AML patients as compared to non-t(8;21) FAB subtype M2 AML and to CD34+ cells in healthy controls. One such gene that was consistently significantly downregulated in t(8;21)+ patients was Ras-association domain family member 2 (RASSF2). RASSF2 is a putative tumor suppressor that is capable of mediating apoptosis (in a Ras dependent manner) through its interactions with the MST1/2 kinases and the cancer-specific apoptotic protein Par-4. RASSF2 has previously been shown to be frequently downregulated via hypermethylation in a wide variety of solid tumors, however little is known about its function in leukemia. Here we demonstrate that RASSF2 is a potentially interesting target for downregulation by the RUNX1-ETO fusion protein. Gene expression analysis by RT-qPCR in leukemia cell lines confirmed that RASSF2 is significantly downregulated in both Kasumi-1 and SKNO t(8;21)+ cell lines as compared to a similar non-t(8;21) HL-60 line. We found that exogenous expression of AML1-ETO in HL-60 leukemia cells induces a rapid downregulation of RASSF2, further supporting that it is a target of this leukemogenic fusion protein. Over-expression of RASSF2 in leukemia cells significantly inhibits their proliferative capability, indicating an important biological effect of RASSF2 in blood cells. Finally, over-expression of RASSF2 significantly inhibits the long-term self-renewal capability of RUNX1-ETO expressing hematopoietic cells as measured by their serial replating ability in a colony formation assay. Discussion Based on the analysis of patient data and our own experiments it appears that RASSF2 is a direct target for downregulation by the AML1-ETO fusion protein. Due to its potential involvement as a mediator of apoptosis in important oncogenic signaling pathways RASSF2 is a strong candidate for further investigation in the context of t(8;21)+ AML pathogenesis. In particular, it will be interesting to continue to investigate the relationship between RASSF2 and apoptotic protein Par-4, as several lines of evidence suggest Par-4 to be therapeutically relevant due to its ability to selectively induce apoptosis in cancer cells. Disclosures: No relevant conflicts of interest to declare.

Beschreibung: RUNX1, also known as AML1, is a DNA binding transcription factor that is expressed in hematopoietic stem and progenitor cells (HSPCs). As demonstrated by several mouse models, RUNX1 is necessary for definitive hematopoiesis and proper homeostasis of HSPCs. Furthermore, mutations of RUNX1have been implicated in patients with a variety of blood-related malignancies and disorders. These findings have established RUNX1 as a master regulator of hematopoiesis. As a transcription factor, RUNX1 exerts its function in hematopoiesis by binding to regulatory regions in order to guide the expression of its direct target genes. Most confirmed RUNX1 target genes are mainly expressed in differentiated blood cells. Direct targets of RUNX1 in HSPCs, however, have largely remained unexplored. Identifying direct target genes of RUNX1 offers an insightful view of how this master regulator influences HSPC function. To elucidate RUNX1 target genes in HSPCs, we have analyzed gene expression signatures from wildtype and RUNX1-deficient HSPCs (Lineage-/cKit+/Sca1+) in a previous report (Matsuura et al., Blood, 2012). With the goal of continuing the characterization of RUNX1 target genes, in this current study, we performed genome-occupancy analysis with chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) using RUNX1 antibodies and a murine HSPC cell line. Bioinformatics analysis of the ChIP-seq data revealed 6370 significant RUNX1 binding peaks (