ALBERT

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: Abstract 3163 The t(8;21)(q22;q22) translocation is one of the most common chromosomal translocations in de novo acute myeloid leukemia (AML). The 8;21 translocation is often associated with additional cytogenetic abnormalities. The loss of the sex chromosome (LOS) is by far the most frequent abnormality found in association with the t(8;21) leukemia, accounting for 32–59% of patients, in contrast to other types of AML in which the LOS occurs in less than 5% of patients. To evaluate the role of sex chromosome deletion in t(8;21)-related leukemogenesis, hematopoietic cells from a mouse line with only one sex chromosome were used in retrovirus-mediated t(8;21) (AML1-ETO) expression and transplantation assays. The absence of leukemia in those animals suggested that a gene present in the pseudoautosomal region of sex chromosomes in humans but not in mice may be the target gene in LOS. The granulocyte-macrophage colony-stimulating factor receptor α (GM-CSFRα) gene is one such gene and is also known to be involved in myeloid cell survival, proliferation and differentiation. The GM-CSFRα gene is specifically down-regulated in AML patients with t(8;21), but not in other common translocations (Valk PJM et al, NEJM, 2004). The GM-CSFR complex is composed of α and βc subunits that assemble into a complex for receptor activation and signaling. To investigate the role of GM-CSFR signaling in t(8;21)-mediated leukemogenesis, GM-CSFR common β subunit knockout (GM-CSFRβc-/-) mice were used in our studies as a model for deficient GM-CSFR signaling. Transduction of AML1-ETO in hematopoietic cells from GM-CSFRβc-/- resulted in myeloid leukemia of a median survival time of 225 days, high percentage of blasts in peripheral blood and bone marrow, anemia, thrombocytopenia, hepatomegaly and splenomegaly. Comparison of wild-type and GM-CSFRβc-/- cells in the same transplantation resulted in development of AML1-ETO-induced leukemia at higher penetrance in GM-CSFRβc-/- cells (28.5% vs 100%). Moreover, the latency of leukemia was shorter in GM-CSFRβc-/- cells than in wild-type cells after transduction of AML1-ETO9a. Analysis of the hematopoietic compartment of healthy GM-CSFRβc-/- mice detected no significant abnormalities in the immature hematopoietic compartment (LSK, CMP, GMP, MEP), suggesting that AML1-ETO expression is required for leukemia to occur. In vitro, expression of AML1-ETO alone is sufficient for the immortalization of normal hematopoietic cells, as demonstrated by serial replating capacity of cells in methylcellulose colony assay. Addition of mGM-CSF to the basic cytokine cocktail (mIL-3, hIL-6, mSCF, hEPO) did not significantly affect number, type, size, and cell composition of colony cells. In contrast, the addition of mGM-CSF eliminated the replating capacity of AML1-ETO expressing cells, although they survived longer than control vector-infected cells. The results suggest that activation of GM-CSF signaling can specifically abrogate the self-renewal ability of potential leukemic stem cells in the early immortalization phase. These results support a possible tumor suppressor role of GM-CSF in leukemogeneis by AML1-ETO and may provide clues to understand how AML1-ETO corrupts normal GM-CSF signals to its own advantage for leukemogenic transformation. Disclosures: No relevant conflicts of interest to declare.

Beschreibung: Abstract 110 While characterizing AML1-ETO domains important for leukemia development and identifying proteins interacting with these domains, we discovered SON as a novel AML1-ETO binding protein (PNAS, 2008, 105:17103). SON is a large, poorly characterized serine/arginine rich SR protein localized to nuclear speckles. SON has DNA, single stranded RNA, and double stranded RNA binding domains and long repeats of amino acids. Overexpression of a partial fragment of SON in a transformed cell line decreased the tumorigenic potential of the cell in nude mice and protected yeast from apoptosis. However, partially due to its large size, the SON protein has not been well characterized. Recently, we reported that SON plays an important role in RNA splicing of a specific set of cell cycle related genes: ones that possess weak splice sites (Molecular Cell, 2011, 42:185). While SON is expressed ubiquitously, its expression level is noticeably higher in hematopoietic organs/tissues and blood cells compared to other tissues, suggesting important roles of SON in the hematopoietic system. To examine whether SON expression is regulated during hematopoietic differentiation, we measured relative mRNA level of mouse Son in different stages of hematopoiesis. Son mRNA level is higher in lineage marker negative (Lin-) bone marrow cells when compared to total bone marrow cells. Macrophages showed less expression of Son, suggesting that Son is down-regulated along the hematopoietic differentiation. We further sorted Lin- cells and measured the Son level in LSK (Lin-, Sca1+, cKit+), CMP (commom myeloid progenitors), GMP (granulocyte/monocyte progenitors) and MEP (megakaryocyte/erythroid progenitors) populations. LSK cells, which precede other progenitors, showed the highest level of Son. In addition, we confirmed that SON is down-regulated during TPA-induced monocytic differentiation of U937 myeloid cells. Taken together, SON is more abundantly expressed in immature hematopoietic cells and down-regulated during differentiation. Since SON is differentially expressed during hematopoietic differentiation, we examined whether SON is involved in regulation of hematopoietic transcription factors that are key dictators of hematopoietic differentiation. Among the several transcription factors analyzed, we found that Gata-2 mRNA was consistently reduced by two different Son shRNAs in Lin- bone marrow cells. Down regulation of the GATA-2 mRNA level was further confirmed in human K562 leukemic cell line. More interestingly, while having 20∼40% reduction of mRNA level, the GATA-2 protein level is more remarkably down-regulated upon SON knockdown, resulting in 75∼90% reduction. These results indicate that upon SON knockdown, GATA-2 protein level is mainly regulated at the post-transcriptional steps. Sequence analysis of the 3' UTR of the human GATA-2 gene predicted several candidates of targeting microRNAs. Among them, we confirmed that the mature form of miR-27a and miR-24 are up-regulated upon SON knockdown. Next, we tested the effect of over-expression of miR-27a and miR-24 on GATA-2 expression using a GATA-2 3' UTR-containing luciferase reporter construct and demonstrated that miR-27a indeed inhibits GATA-2 mRNA level. miR-27a is a member of the miR-23a∼27a∼24-2 cluster. RTqPCR showed that primary miRNA of the miR-23a∼27a∼24 cluster is upregulated upon SON knockdown. To test whether the increase of pri-miR of this cluster is due to promoter activation, we used a reporter construct containing the promoter sequence of the miR-23a∼27a∼24-2 cluster fused to the luciferase reporter. The expression of luciferase driven by this promoter is significantly elevated upon SON knockdown, suggesting that that SON functions to repress transcription of the miR-23a-27a-24-2 cluster, thereby relieving GATA-2 mRNA from targeting by miR-27a, and contributes to maintaining the GATA-2 protein level. Taken together, our results reveal a previously unidentified function of SON in microRNA transcription and controlling the GATA-2 protein level in hematopoietic cells. Disclosures: No relevant conflicts of interest to declare.

Beschreibung: RUNX1 is the transcription factor described as the master regulator of hematopoiesis. Due to its central role during blood development, numerous RUNX1 mutations have been reported in hematologic abnormalities. Mice null for Runx1 die during embryogenesis, lacking definitive HSCs. Conditional Runx1Δ/Δ mice are viable, but exhibit a variety of blood abnormalities. The most salient defect in these Runx1Δ/Δ mice is expansion of the hematopoietic stem and progenitor cell (HSPC) population, measured as an increase in number of lineage negative, Sca1 positive, cKit positive (LSK) cells. A shortened form of RUNX1 (RUNX1SF) lacking the C-terminal and part of the N-terminal domain (41-214) acts as a dominant negative regulator of RUNX1 and hence also models RUNX1 loss-of-function. A differential gene expression analysis of HSPCs derived from Runx1Δ/Δ compared to wild type mice uncovered GTPase immunity-associated protein family member 4 (GIMAP4) as one of the genes most highly upregulated. Previous studies have focused almost exclusively on the role of GIMAP4 as a pro-apoptotic protein during T-cell development. This study illuminates a novel non-apoptotic role of GIMAP4 in a formerly unstudied HSPC context. Runx1Δ/Δ mice were crossed with Gimap4-/- mice to generate a double knockout (dKO) mouse line. These dKO mice exhibited attenuated HSPC proliferation in comparison to Runx1Δ/Δ mice, suggesting that GIMAP4 functions in this HSPC expansion phenotype. BMT experiments using lethally irradiated C57 mice and RUNX1SF transduced wild type versus Gimap4-/-bone marrow confirmed this result. GIMAP4 also worked independently and coordinately with RUNX1 to influence individual progenitor populations. Common lymphoid progenitors (CLP) were affected only by GIMAP4. Gimap4-/- mice exhibited an expansion of the CLP population, consistent with its pro-apoptotic role in lymphoid populations. Conversely, both RUNX1 and GIMAP4 coordinately exerted an effect on myeloid progenitor populations. Runx1Δ/Δ mice harbored expanded granulocyte-macrophage progenitor (GMP) and common myeloid progenitor (CMP) populations. This expansion was not observed when GIMAP4 was also ablated. This suggests a pro-proliferative role of GIMAP4 specifically in myeloid populations. These opposing roles of GIMAP4 in lymphoid versus myeloid cells suggest a more contextual, cell-specific role of this GTPase protein. Ultimately, this study provides insight into how RUNX1 and GIMAP4 may coordinate to maintain HSPC homeostasis. Disclosures No relevant conflicts of interest to declare.

Beschreibung: The stem cell factor (SCF)/Kit system has served as a classic model in deciphering molecular signaling events in the hematopoietic compartment, and Kit expression is a most critical marker for hematopoietic stem cells (HSCs) and progenitors. However, it remains to be elucidated how Kit expression is regulated in HSCs. Herein we report that a cytoplasmic tyrosine phosphatase Shp2, acting downstream of Kit and other RTKs, promotes Kit gene expression, constituting a Kit-Shp2-Kit signaling axis. Inducible ablation of PTPN11/Shp2 resulted in severe cytopenia in BM, spleen, and peripheral blood in mice. Shp2 removal suppressed the functional pool of HSCs/progenitors, and Shp2-deficient HSCs failed to reconstitute lethally irradiated recipients because of defects in homing, self-renewal, and survival. We show that Shp2 regulates coordinately multiple signals involving up-regulation of Kit expression via Gata2. Therefore, this study reveals a critical role of Shp2 in maintenance of a functional HSC/progenitor pool in adult mammals, at least in part through a kinase-phosphatase-kinase cascade.

Beschreibung: Chromosome translocation 8q22;21q22 [t(8;21)] is commonly associated with acute myeloid leukemia (AML), and the resulting AML1-ETO fusion proteins are involved in the pathogenesis of AML. To identify novel molecular and therapeutic targets, we performed combined gene expression microarray and promoter occupancy (ChIP-chip) profiling using Lin−/Sca1−/cKit+ cells, the major leukemia cell population, from an AML mouse model induced by AML1-ETO9a (AE9a). Approximately 30% of the identified common targets of microarray and ChIP-chip assays overlap with the human t(8;21)–gene expression molecular signature. CD45, a protein tyrosine phosphatase and a negative regulator of cytokine/growth factor receptor and JAK/STAT signaling, is among those targets. Its expression is substantially down-regulated in leukemia cells. Consequently, JAK/STAT signaling is enhanced. Re-expression of CD45 suppresses JAK/STAT activation, delays leukemia development, and promotes apoptosis of t(8;21)–positive cells. This study demonstrates the benefit of combining gene expression and promoter occupancy profiling assays to identify molecular and potential therapeutic targets in human cancers and describes a previously unappreciated signaling pathway involving t(8;21) fusion proteins, CD45, and JAK/STAT, which could be a potential novel target for treating t(8;21) AML.

Beschreibung: Abstract 347 Both human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) are pluripotent stem cells (hPSCs) with potential to differentiate into all types of somatic cells. Patients suffering from blood disorders can be cured with hematopoietic cell transplantations (HCT). Technical advancements in hPSC production and handling have revolutionized their potential applications in regenerative medicine and provided enormous hope for patients who may need HCT. hiPSCs derived from autologous cells could provide unlimited leukocyte antigen matched blood cells on a patient-specific basis. A remaining hurdle in this process remains the need for efficient and effective generation of specific blood cells from hPSCs for therapeutic use. Transcription factors play key roles in regulating maintenance, expansion, and differentiation of blood cells from hPSCs. Studies have shown that transcription factor RUNX1 is required for the formation of definitive blood cells. There are several alternatively spliced isoforms of the RUNX1 protein, including the shortest form RUNX1a and two longer forms RUNX1b and RUNX1c. Based on known properties of RUNX1 proteins, we hypothesized that RUNX1a promotes the production of therapeutic hematopoietic stem cells from hPSCs. By employing ectopic expression of RUNX1a on different human ESC and iPSC lines (H9, BC1, iCB5) under a defined hematopoietic differentiation system, we aimed to identify function of RUNX1a on lineage commitment and molecular mechanisms of RUNX1 activity in differentiation of PSCs to hematopoietic cells. We demonstrated that expression of endogenous RUNX1a parallels lineage commitment and hematopoietic emergence from hPSCs. During differentiation process RUNX1a enhanced the expression of several mesoderm and hematopoietic differentiation related factors, including KDR, SCL, GATA2, and PU.1. In addition, over-expression of RUNX1a in embryoid bodies (EBs) showed more efficient and earlier emergence of typical sac structures, which predicts cell lineage commitment and germ layer development at the early stage of EB differentiation. At day 7, EBs derived from hPSCs was dissociated into single cells for flow cytometry analysis. The mean frequency of CD31+CD34+CD45− and total CD34+ cells with hemato-endothelial cell features are 35.1% and 67.1% from RUNX1a-overexpressing EBs, and 8.7% and 24.1% from vector control EBs. Immunohistochemistry analysis of EBs at day 9 of differentiation confirmed that expression of RUNX1a accelerated mesoderm commitment and emergence of hemato-endothelial precursors. Flow cytometry analysis on EBs collected at days 9, 11, 13 showed that ectopic RUNX1a induced a robust increase in the frequency of hematopoietic progenitor cells in all hPSC lines examined. At Day 9, RUNX1a-overexpressing EBs generated 48.5% CD43+CD45+ cells, 45.1% CD34+CD45+ cells, and 8.5 folds higher CD43+ cells than vector EBs. Later at Day 13, 80% CD45+ and 75% CD43+/CD34+CD45+ hematopoietic stem/progenitor cells (HSPCs) achieved from dissociated EBs. In liquid culture, RUNX1a HSPC showed strong expansion and high percentage of CD235a+CD45− (20%) and CD71+CD235a+ (16%), markers for erythroid populations. Flow cytometry and western blots on RUNX1a-EB formed colonies showed significantly higher β-globin production than that of the vector, suggesting expression of RUNX1a in HSPC enhanced definitive hematopoiesis. RUNX1a-hPSCs derived HSPCs possess self-renewal capability and are capable of differentiating into multi-lineages ex vivo. Furthermore HSPCs generated from RUNX1a-EBs possessed the capacity of interacting with surrogate niche and showed long-term repopulation ability under LTC-IC (Long-Term Culture-Initiating Cell Assay) condition. Colonies generated from HSPC of RUNX1a-EBs after 3 week bulk LTC-IC culture showed 300 folds higher than vector control. RUNX1a-hPSCs derived CD34+CD45+ cells could maintain a non-adherent population in ouldCD45+ sEBsND THIS SENTENCE5 week culture on stromal cell M210. In summary we identified that RUNX1a enhances derivation of definitive hematopoietic cells from human PSCs. Our study provides an important and useful system to enhance specificity and efficiency of generating functional blood cells and further differentiated cells from human PSCs, which may provide valuable source for future clinical applications in patients with hematologic disorders. 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: Fusion protein AML1-ETO, resulting from t(8;21) translocation, is highly related to leukemia development. It has been reported that full-length AML1-ETO blocks AML1 function and requires additional mutagenic events to promote leukemia. We have previously shown that the expression of AE9a, a splice isoform of AML1-ETO, can rapidly cause leukemia in mice. To understand how AML1-ETO is involved in leukemia development, we took advantage of our AE9a leukemia model and sought to identify its interacting proteins from primary leukemic cells. Here, we report the discovery of a novel AE9a binding partner PRMT1 (protein arginine methyltransferase 1). PRMT1 not only interacts with but also weakly methylates arginine 142 of AE9a. Knockdown of PRMT1 affects expression of a specific group of AE9a-activated genes. We also show that AE9a recruits PRMT1 to promoters of AE9a-activated genes, resulting in enrichment of H4 arginine 3 methylation, H3 Lys9/14 acetylation, and transcription activation. More importantly, knockdown of PRMT1 suppresses the self-renewal capability of AE9a, suggesting a potential role of PRMT1 in regulating leukemia development.

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.