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The ZEB2 transcription factor has been demonstrated to play important roles in hematopoiesis and leukemic transformation. ZEB1 is a close family member of ZEB2 but has remained more enigmatic concerning its roles in hematopoiesis. Here, we show using conditional loss-of-function approaches and bone marrow (BM) reconstitution experiments that ZEB1 plays a cell-autonomous role in hematopoietic lineage differentiation, particularly as a positive regulator of monocyte development in addition to its previously reported important role in T-cell differentiation. Analysis of existing single-cell (sc) RNA sequencing (RNA-seq) data of early hematopoiesis has revealed distinctive expression differences between Zeb1 and Zeb2 in hematopoietic stem and progenitor cell (HSPC) differentiation, with Zeb2 being more highly and broadly expressed than Zeb1 except at a key transition point (short-term HSC [ST-HSC]➔MPP1), whereby Zeb1 appears to be the dominantly expressed family member. Inducible genetic inactivation of both Zeb1 and Zeb2 using a tamoxifen-inducible Cre-mediated approach leads to acute BM failure at this transition point with increased long-term and short-term hematopoietic stem cell numbers and an accompanying decrease in all hematopoietic lineage differentiation. Bioinformatics analysis of RNA-seq data has revealed that ZEB2 acts predominantly as a transcriptional repressor involved in restraining mature hematopoietic lineage gene expression programs from being expressed too early in HSPCs. ZEB1 appears to fine-tune this repressive role during hematopoiesis to ensure hematopoietic lineage fidelity. Analysis of Rosa26 locus–based transgenic models has revealed that Zeb1 as well as Zeb2 cDNA-based overexpression within the hematopoietic system can drive extramedullary hematopoiesis/splenomegaly and enhance monocyte development. Finally, inactivation of Zeb2 alone or Zeb1/2 together was found to enhance survival in secondary MLL-AF9 acute myeloid leukemia (AML) models attesting to the oncogenic role of ZEB1/2 in AML., This study shows that the closely related transcription factors ZEB1 and ZEB2 cooperate to restrain myeloid and lymphoid differentiation programs in hematopoietic stem and progenitor cells, ensuring fidelity of differentiation in multiple lineages.

Acute erythroleukemia (AEL or acute myeloid leukemia [AML]-M6) is a rare but aggressive hematologic malignancy. Previous studies showed that AEL leukemic cells often carry complex karyotypes and mutations in known AML-associated oncogenes. To better define the underlying molecular mechanisms driving the erythroid phenotype, we studied a series of 33 AEL samples representing 3 genetic AEL subgroups including TP53-mutated, epigenetic regulator-mutated (eg, DNMT3A, TET2, or IDH2), and undefined cases with low mutational burden. We established an erythroid vs myeloid transcriptome-based space in which, independently of the molecular subgroup, the majority of the AEL samples exhibited a unique mapping different from both non-M6 AML and myelodysplastic syndrome samples. Notably, >25% of AEL patients, including in the genetically undefined subgroup, showed aberrant expression of key transcriptional regulators, including SKI, ERG, and ETO2. Ectopic expression of these factors in murine erythroid progenitors blocked in vitro erythroid differentiation and led to immortalization associated with decreased chromatin accessibility at GATA1-binding sites and functional interference with GATA1 activity. In vivo models showed development of lethal erythroid, mixed erythroid/myeloid, or other malignancies depending on the cell population in which AEL-associated alterations were expressed. Collectively, our data indicate that AEL is a molecularly heterogeneous disease with an erythroid identity that results in part from the aberrant activity of key erythroid transcription factors in hematopoietic stem or progenitor cells.

International audience; Chimeric transcription factors are a hallmark of human leukemia, but the molecular mechanisms by which they block differentiation and promote aberrant self-renewal remain unclear. Here, we demonstrate that the ETO2-GLIS2 fusion oncoprotein, which is found in aggressive acute megakaryoblastic leukemia, confers megakaryocytic identity via the GLIS2 moiety while both ETO2 and GLIS2 domains are required to drive increased self-renewal properties. ETO2-GLIS2 directly binds DNA to control transcription of associated genes by upregulation of expression and interaction with the ETS-related ERG protein at enhancer elements. Importantly, specific interference with ETO2-GLIS2 oligomerization reverses the transcriptional activation at enhancers and promotes megakaryocytic differentiation, providing a relevant interface to target in this poor-prognosis pediatric leukemia.

First reported in 1999, germline runt-related transcription factor 1 (RUNX1) mutations are a well-established cause of familial platelet disorder with predisposition to myeloid malignancy (FPD-MM). We present the clinical phenotypes and genetic mutations detected in 10 novel RUNX1-mutated FPD-MM families. Genomic analyses on these families detected 2 partial gene deletions, 3 novel mutations, and 5 recurrent mutations as the germline RUNX1 alterations leading to FPD-MM. Combining genomic data from the families reported herein with aggregated published data sets resulted in 130 germline RUNX1 families, which allowed us to investigate whether specific germline mutation characteristics (type, location) could explain the large phenotypic heterogeneity between patients with familial platelet disorder and different HMs. Comparing the somatic mutational signatures between the available familial (n = 35) and published sporadic (n = 137) RUNX1-mutated AML patients showed enrichment for somatic mutations affecting the second RUNX1 allele and GATA2. Conversely, we observed a decreased number of somatic mutations affecting NRAS, SRSF2, and DNMT3A and the collective genes associated with CHIP and epigenetic regulation. This is the largest aggregation and analysis of germline RUNX1 mutations performed to date, providing a unique opportunity to examine the factors underlying phenotypic differences and disease progression from FPD to MM.

Modulators of epithelial-to-mesenchymal transition (EMT) have recently emerged as novel players in the field of leukemia biology. The mechanisms by which EMT modulators contribute to leukemia pathogenesis, however, remain to be elucidated. Here we show that overexpression of SNAI1, a key modulator of EMT, is a pathologically relevant event in human acute myeloid leukemia (AML) that contributes to impaired differentiation, enhanced self-renewal, and proliferation of immature myeloid cells. We demonstrate that ectopic expression of Snai1 in hematopoietic cells predisposes mice to AML development. This effect is mediated by interaction with the histone demethylase KDM1A/LSD1. Our data shed new light on the role of SNAI1 in leukemia development and identify a novel mechanism of LSD1 corruption in cancer. This is particularly pertinent given the current interest surrounding the use of LSD1 inhibitors in the treatment of multiple different malignancies, including AML.

Acute erythroid leukemia (AEL) is a high-risk leukemia of poorly understood genetic basis, with controversy regarding diagnosisin the spectrum of myelodysplasia and myeloid leukemia. We compared genomic features of 159 childhood and adult AEL cases with non-AEL myeloid disorders and defined five age-related subgroups with distinct transcriptional profiles: adult, TP53mutated; NPM1 mutated; KMT2A mutated/rearranged; adult, DDX41 mutated; and pediatric, NUP98 rearranged. Genomic features influenced outcome, with NPM1 mutations and HOXB9 overexpression being associated with a favorable prognosis andTP53, FLT3 or RB1 alterations associated with poor survival. Targetable signaling mutations were present in 45% of cases and included recurrent mutations of ALK and NTRK1, the latter of which drives erythroid leukemogenesis sensitive to TRK inhibition.This genomic landscape of AEL provides the framework for accurate diagnosis and risk stratification of this disease, and the rationale for testing targeted therapies in this high-risk leukemia. Refereed/Peer-reviewed

Nanomedicine holds great promise for vascular disease diagnosis and specific therapy, yet rapid sequestration by the mononuclear phagocytic system limits the efficacy of particle-based agents. The use of low-fouling polymers, such as poly(ethylene glycol), efficiently reduces this immune recognition, but these nondegradable polymers can accumulate in the human body and may cause adverse effects after prolonged use. Thus, new particle formulations combining stealth, low immunogenicity and biocompatible features are required to enable clinical use. Here, a low-fouling particle platform is described using exclusively protein material. A recombinant protein with superior hydrophilic characteristics provided by the amino acid repeat proline, alanine, and serine (PAS) is designed and cross-linked into particles with lysine (K) and polyglutamic acid (E) using mesoporous silica templating. The obtained PASKE particles have low-fouling behavior, have a prolonged circulation time compared to albumin-based particles, and are rapidly degraded in the cell's lysosomal compartment. When labeled with near-infrared fluorescent molecules and functionalized with an anti-glycoprotein IIb/IIIa single-chain antibody targeting activated platelets, the particles show potential as a noninvasive molecular imaging tool in a mouse model of carotid artery thrombosis. The PASKE particles constitute a promising biodegradable and versatile platform for molecular imaging of vascular diseases.

Aberrant expression of the SNAIL & ZEB families of Epithelial to Mesenchymal Transition (EMT) modulators is an emerging theme in acute leukemia biology. EMT regulation has long been implicated in epithelial tumor pathogenesis, however a role in hematopoietic malignancy has only recently been explored. In this study, we describe a novel mechanism of AML pathogenesis involving SNAI1 induced epigenetic dysregulation via corruption of the histone demethylase LSD1. SNAI1 is expressed ∼10-fold higher in AML blasts compared to normal hematopoietic cells, suggesting a functional role for SNAI1 in AML. Indeed, SNAI1 knockdown induces differentiation in human AML cells, while Snai1 deletion delays tumour onset in mouse AML models. Furthermore, transgenic Snai1 expression perturbs myeloid development, resulting in accumulation of immature myeloid cells with enhanced self-renewal capacity - ultimately predisposing mice to AML. Intriguingly, impaired myeloid development by Snai1 absolutely requires its interaction with Lsd1, a H3K4 histone demethylase that has emerged as a novel therapeutic target in AML. Mechanistically, this interaction results in corruption of Lsd1 function via aberrant recruitment to Snai1 gene targets, and sequestration away from its own targets. Ultimately, this leads to changes in H3K4me1/2 methylation at specific promoters and enhancers, and subsequently altered gene expression. This model provides insight into the mechanisms by which LSD1 can be perturbed in AML, and offers a possible explanation for why LSD1 inhibition/loss has somewhat dichotomous effects on normal and malignant hematopoietic stem cells. Furthermore, as other members of the SNAIL and ZEB families are known to interact with LSD1, it is likely that this model will be more broadly applicable to other EMT modulators in AML, representing a possible new focus for future therapeutic studies.

Elevated expression of the Zinc finger E-box binding homeobox transcription factor-2 (ZEB2) is correlated with poor prognosis and patient outcome in a variety of human cancer subtypes. Using a conditional gain-of-function mouse model, we recently demonstrated that ZEB2 is an oncogenic driver of immature T-cell acute lymphoblastic leukemia (T-ALL), a heterogenic subgroup of human leukemia characterized by a high incidence of remission failure or hematological relapse after conventional chemotherapy. Here, we identified the lysine-specific demethylase KDM1A as a novel interaction partner of ZEB2 and demonstrated that mouse and human T-ALLs with increased ZEB2 levels critically depend on KDM1A activity for survival. Therefore, targeting the ZEB2 protein complex through direct disruption of the ZEB2-KDM1A interaction or pharmacological inhibition of the KDM1A demethylase activity itself could serve as a novel therapeutic strategy for this aggressive subtype of human leukemia and possibly other ZEB2-driven malignancies.

Snail family proteins are key inducers of the epithelial-mesenchymal transition (EMT), a critical process required for normal embryonic development. They have also been strongly implicated in regulating the EMT-like processes required for tumour cell invasion, migration, and metastasis. Whether these proteins also contribute to normal blood cell development, however, remains to be clearly defined. Increasing evidence supports a role for the Snail family in regulating cell survival, migration, and differentiation within the haematopoietic system, as well as potentially an oncogenic role in the malignant transformation of haematopoietic stem cells. This review will provide a broad overview of the Snail family, including key aspects of their involvement in the regulation and development of solid organ cancer, as well as a discussion on our current understanding of Snail family function during normal and malignant haematopoiesis.

We report the discovery of GATA2 as a new myelodysplastic syndrome (MDS)-acute myeloid leukemia (AML) predisposition gene. We found the same, previously unidentified heterozygous c.1061C>T (p.Thr354Met) missense mutation in the GATA2 transcription factor gene segregating with the multigenerational transmission of MDS-AML in three families and a GATA2 c.1063_1065delACA (p.Thr355del) mutation at an adjacent codon in a fourth MDS family. The resulting alterations reside within the second zinc finger of GATA2, which mediates DNA-binding and protein-protein interactions. We show differential effects of the mutations on the transactivation of target genes, cellular differentiation, apoptosis and global gene expression. Identification of such predisposing genes to familial forms of MDS and AML is critical for more effective diagnosis and prognosis, counseling, selection of related bone marrow transplant donors and development of therapies. Refereed/Peer-reviewed

Platelets are the small anuclear blood cells that are the product of megakaryocytopoiesis, the process of hematopoietic stem cell commitment to megakaryocyte production and the differentiation and maturation of these cells for platelet release. Deregulation or disruption of megakaryocytopoiesis can result in platelet deficiencies, the thrombocytopenias, with attendant risk of hemorrhage or thrombocytosis, a pathological excess of platelet numbers. Mouse models, particularly those engineered to carry genetic alterations modeling mutations associated with human disease, have provided important insights into megakaryocytopoiesis and deregulation of this process in disease. This review focuses on mouse models of diseases of altered megakaryocyte and platelet number, illustrating the profound contribution of these models in validating suspected roles of disease-associated genetic alterations, promoting discovery of new links between genetic mutations and specific diseases, and providing unique tools for better understanding of disease pathophysiology and progression, as well as resources to define drug action or develop new therapeutic strategies.

Summary Background: RNA interference (RNAi) is a powerful tool for suppressing gene function. The tetracycline (tet)-regulated expression system has recently been adapted to allow inducible RNAi in mice, however its efficiency in a particular cell type in vivo depends on a transgenic tet transactivator expression pattern and is often highly variable. Objective: We aimed to establish a transgenic strategy that allows efficient and inducible gene knockdown in particular hematopoietic lineages in mice. Methods and results: Using a tet-regulated reporter gene strategy, we found that transgenic mice expressing the rtTA (tet-on) transactivator under control of the cytomegalovirus (CMV) promoter (CMV-rtTA mice) display inducible reporter gene expression with unusual and near-complete efficiency in megakaryocytes and platelets. To test whether the CMV-rtTA transgene can drive inducible and efficient gene knockdown within this lineage, we generated a novel mouse strain harboring a tet-regulated short hairpin RNA (shRNA) targeting Bcl-x(L) , a pro-survival Bcl-2 family member known to be essential for maintaining platelet survival. Doxycycline treatment of adult mice carrying both transgenes induces shRNA expression, depletes Bcl-x(L) in megakaryocytes and triggers severe thrombocytopenia, whereas doxycycline withdrawal shuts off shRNA expression, normalizes Bcl-x(L) levels and restores platelet numbers. These effects are akin to those observed with drugs that target Bcl-x(L) , clearly demonstrating that this transgenic system allows efficient and inducible inhibition of genes in megakaryocytes and platelets. Conclusions: We have established a novel transgenic strategy for inducible gene knockdown in megakaryocytes and platelets that will be useful for characterizing genes involved in platelet production and function in adult mice.

Down syndrome is characterized by multiple phenotypic manifestations associated with trisomy of chromosome 21. The transient myeloproliferative disorder and acute megakaryocytic leukemia associated with Down syndrome are uniquely associated with mutations in the transcription factor GATA1; however, the identity of trisomic genes on chromosome 21 that predispose to these hematologic disorders remains unknown. Using a loss-of-function allele, we show that specific reduction to functional disomy of the Erg gene corrects the pathologic and hematologic features of myeloproliferation in the Ts(1716)65Dn mouse model of Down syndrome, including megakaryocytosis and progenitor cell expansion. Our data provide genetic evidence establishing the need for Erg trisomy for myeloproliferation in Ts(1716)65Dn mice and imply that increased ERG gene dosage may be a key consequence of trisomy 21 that can predispose to malignant hematologic disorders in Down syndrome.

Background The presence of histone 3 lysine 9 (H3K9) methylation on the mouse inactive X chromosome has been controversial over the last 15 years, and the functional role of H3K9 methylation in X chromosome inactivation in any species has remained largely unexplored. Results Here we report the first genomic analysis of H3K9 di- and tri-methylation on the inactive X: we find they are enriched at the intergenic, gene poor regions of the inactive X, interspersed between H3K27 tri-methylation domains found in the gene dense regions. Although H3K9 methylation is predominantly non-genic, we find that depletion of H3K9 methylation via depletion of H3K9 methyltransferase Set domain bifurcated 1 (Setdb1) during the establishment of X inactivation, results in failure of silencing for around 150 genes on the inactive X. By contrast, we find a very minor role for Setdb1-mediated H3K9 methylation once X inactivation is fully established. In addition to failed gene silencing, we observed a specific failure to silence X-linked long-terminal repeat class repetitive elements. Conclusions Here we have shown that H3K9 methylation clearly marks the murine inactive X chromosome. The role of this mark is most apparent during the establishment phase of gene silencing, with a more muted effect on maintenance of the silent state. Based on our data, we hypothesise that Setdb1-mediated H3K9 methylation plays a role in epigenetic silencing of the inactive X via silencing of the repeats, which itself facilitates gene silencing through alterations to the conformation of the whole inactive X chromosome. Electronic supplementary material The online version of this article (doi:10.1186/s13072-016-0064-6) contains supplementary material, which is available to authorized users.

To define genetic lesions driving leukemia, we targeted cre-dependent Sleeping Beauty (SB) transposon mutagenesis to the blood-forming system using a hematopoietic-selective vav 1 oncogene ( vav1 ) promoter. Leukemias of diverse lineages ensued, most commonly lymphoid leukemia and erythroleukemia. The inclusion of a transgenic allele of Janus kinase 2 ( JAK2 ) V617F resulted in acceleration of transposon-driven disease and strong selection for erythroleukemic pathology with transformation of bipotential erythro-megakaryocytic cells. The genes encoding the E-twenty-six (ETS) transcription factors Ets related gene (Erg) and Ets1 were the most common sites for transposon insertion in SB-induced JAK2V617F -positive erythroleukemias, present in 87.5% and 65%, respectively, of independent leukemias examined. The role of activated Erg was validated by reproducing erythroleukemic pathology in mice transplanted with fetal liver cells expressing translocated in liposarcoma ( TLS ) -ERG , an activated form of ERG found in human leukemia. Via application of SB mutagenesis to TLS-ERG –induced erythroid transformation, we identified multiple loci as likely collaborators with activation of Erg . Jak2 was identified as a common transposon insertion site in TLS-ERG –induced disease, strongly validating the cooperation between JAK2V617F and transposon insertion at the Erg locus in the JAK2V617F -positive leukemias. Moreover, loci expressing other regulators of signal transduction pathways were conspicuous among the common transposon insertion sites in TLS-ERG –driven leukemia, suggesting that a key mechanism in erythroleukemia may be the collaboration of lesions disturbing erythroid maturation, most notably in genes of the ETS family, with mutations that reduce dependence on exogenous signals.

Introduction: The genetic basis of several acute myeloid leukemia (AML) subtypes remains poorly characterized, such as that of acute erythroid leukemia (AEL, AML M6) which is currently subclassified by morphology alone, and is associated with poor outcome. Here we sought to perform a definitive genomic analysis of AEL and translate these findings into faithful experimental models and novel therapeutic approaches. Methods: We studied 151 AEL cases (19.2% pediatric, 4.6% young adult, 21.9% adult and 54.3% older adult). Diagnosis of AEL was centrally confirmed and subclassified according to WHO 2008 and revised 2016 criteria. Whole exome and/or genome sequencing, RNA-sequencing and SNP array analysis were performed on all cases and in 2 AEL cell lines (TF-1 and Hel). Genomic data were compared to those from non-M6 childhood and adult AML from TARGET (n=192) and TCGA (n=197) studies. The functional effects of fusion transcripts and mutated genes were examined in IL-3 dependent Ba/F3 cells, NIH3T3 cells for focus formation assays and/or mouse lineage negative hematopoietic stem cells (lin- HSC) for colony forming and transplantation assays. Avatars of human AEL were established in immunocompromised NSGS and MISTRG mice for preclinical studies. Results: a) Genomic landscape of AEL. We identified 2,250 non-synonymous clonal and subclonal somatic mutations in 1,723 genes with a mean of 16.4 per case (range 2-88) and with missense and frameshift mutations accounting for 47.1% and 22.5% of all mutations, respectively. 78 genes were recurrently mutated in at least 3 cases. In frame fusions were detected in 31% of childhood and 27.5% of adult cases, and were more frequent in cases with complex karyotype. 124 potential driver genes were identified by statistical analysis or known pathogenic role in cancer, 9 of which were recurrent novel targets of mutation, most commonly involving chromatin modification (60.3%), cell cycle/tumor suppression (TP53, 33.8%), DNA methylation (28.5%), transcription regulation (26.5%), splicing (15.9%), NPM1 (11.9%), Ras (11.3%), JAK-STAT signaling (9.9%), the cohesin complex (8.6%), ALK/NTRK1 (4.6%) and PI3K signaling (3.3%). Overall, 33% of cases harbored a mutation in signaling genes amenable to inhibition by tyrosine kinase/Ras inhibitors. Mutations in TP53 and DNA methylation genes were significantly more frequent in adults while mutations in transcription regulators and Ras pathway were more frequent in children. Splicing mutations correlated with MDS phenotype and PI3K alterations with therapy-related AEL. Based on the co-occurrence and exclusivity of mutations 7 main distinct AEL genetic subtypes were defined: 1) pediatric AEL with NUP98-rearrangements (3.3% of all cases); 2) adult complex karyotype AEL with TP53 mutations (33.8%); 3) AEL with MLL-rearrangements (12.6%); 4) NPM1-mutated AEL (11.9%); 5) DNA-methylation/splicing mutated AEL (17.8%); 6) splicing/Ras/transcription regulation mutated AEL (21.2%) and 7) Other (8.6%) (Fig.1A). Mutations of chromatin modifiers occurred independently of karyotype, age and subtype (Fig.1B). NUP98-fusions and mutations in PTPN11, UBTF and GATA1 were more frequent in pediatric AEL compared to non-M6 AML. Among adults, mutations in TP53 and MLL were more frequent in AEL while FLT3, NPM1, DNMT3A and IDH1 were more in frequent in non-M6 subtypes. A complex karyotype, therapy-related AEL, TP53 mutations and NUP98-rearrangements were associated with poor outcome. b) Functional AEL modeling and therapeutic translations. Expression of NUP98-JARID1A in lin- HSC resulted in sustained self-renewal and development of an aggressive transplantable leukemia. At least three classes of signaling pathway mutations are targetable in AEL. ALK mutations in the extracellular MAM domain transformed Ba/F3 cells which were sensitive to crizotinib in vitro. Mutations in the tyrosine kinase domain of NTRK1 transformed NIH3T3 cells and were sensitive to entrectinib in vitro. Targeting of JAK-STAT, mTOR and PI3K pathways were examined in xenografts and sensitivity to JAK2 inhibitor ruxolitinib was confirmed in vivo. Conclusions: We provided the first comprehensive landscape of genomic alterations in AEL and defined distinct genomic groups with unique patterns of mutation occurrence compared to non-M6 AML. Finally, we showed that several pathogenic pathways are amenable to inhibition by approved targeted compounds. Disclosures Meggendorfer: MLL Munich Leukemia Laboratory: Employment. Wei:Novartis: Honoraria, Research Funding. Loh:Abbvie: Research Funding; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Mullighan:Amgen: Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees; Loxo Oncology: Research Funding.

Introduction: Regulation of the epithelial to mesenchymal transition (EMT) is an emerging theme in acute leukemia biology (1, 2). EMT is required for many aspects of development, including gastrulation and mesoderm formation. It has also been associated with malignant processes such as epithelial tumor cell invasion and metastasis, and the development, function and chemo-resistance of cancer stem cells. Master regulators of EMT belong to the Snail (Snai1/Snai2/Snai3) and Zeb (Zeb1/Zeb2) families of transcription factors. While they have primarily been studied in relation to solid organ development and embryogenesis, recent work has begun to uncover novel roles for these proteins in both normal and malignant hematopoiesis. A mounting body of evidence implicates the founding member of the Snail family, SNAI1, in Acute Myeloid Leukaemia (AML) development. Transgenic Snai1 expression induces myeloid leukaemia in mice (3), and human AML cells show increased SNAI1 expression compared to haematopoietic stem and progenitor cells (HSPCs). We have also found that shRNA knockdown of SNAI1 in AML cells induces differentiation, as measured by increased CD11b expression. In this paper we elucidate the mechanisms by which elevated levels of Snai1 impair normal hematopoiesis and promote leukemogenesis. Results: We employed murine retroviral and transgenic models to drive high levels of Snai1 in hematopoietic cells in vivo. Homozygous expression of a Snai1 transgene, using the endothelial and hematopoietic restricted Tie2-Cre, resulted in embryonic lethality at E15.5-17.5. Embryos were highly anemic, exhibiting normal primitive erythropoiesis, but severely impaired definitive erythropoiesis. Retroviral mediated overexpression of Snai1 during adult hematopoiesis resulted in a significant expansion of the Granulocyte-Macrophage progenitor (GMP) compartment, with a concomitant reduction in Megakaryocyte-Erythroid progenitor (MEP) numbers. Downstream myeloid development was also perturbed, with a strong differentiation bias towards macrophage production, at the expense of granulocyte development. Mice (n=4) have also begun to develop an aggressive AML beginning at 12 months of age, consistent with the previously published transgenic model (3). The effects of Snai1 expression on myeloid development are reminiscent of those observed in mice lacking Gfi1 or Gfi1b, suggesting possible competition for, or altered function of, their common co-repressor: the chromatin modulator Lsd1. To test this hypothesis, we generated a mutant form of Snai1 that does not bind Lsd1 and retrovirally expressed it in HSPCs. Unlike wild-type Snai1, the mutant form was unable to induce hematopoietic defects or AML development in mice. We next conducted gene expression profiling on the HPC7 hematopoietic progenitor cell line, with or without ectopic Snai1 expression. We observed a significant up-regulation of GMP-associated genes and more mature monocyte/macrophage related genes. These data, along with morphological analysis, suggested that Snai1 expressing HPC7 cells take on a macrophage-biased GMP-like phenotype, but do not fully differentiate into mature myeloid cells. Moreover, there was a significant correlation to gene expression programs from Gfi1/1b knockout HSCs, further implicating loss of Gfi1/1b activity in driving these myeloid phenotypes. Strikingly, we also observed evidence of inflammatory cytokine production and activation of key downstream signaling pathways that contribute to AML cell survival and proliferation. Whether this effect is due to altered regulation of Lsd1 targets, or instead represents a distinct Lsd1-independent mechanism, remains to be determined. Conclusion: We have shown that Snai1 expression perturbs myeloid cell development and induces AML in an Lsd1-dependent manner. Snai1 expression likely contributes to increased survival and proliferation of leukemic cells via induction of key inflammatory signaling pathways that support tumor cell growth. These data confirm the importance of Snai1 in AML development and pathogenesis. References: 1. Goossens S, et al. (2015).Nature communications 6:5794. 2. Stavropoulou V, et al. (2016). Cancer cell 30(1):43-58. 3. Perez-Mancera PA, et al. (2005). Human molecular genetics 14(22):3449-3461. Disclosures No relevant conflicts of interest to declare.

Deregulated gene expression due to genetic alterations, such as gene fusions affecting transcription and/or epigenetic factors is the hallmark of acute myeloid leukemia and the basis for the differentiation block of hematopoietic progenitors. Acute megakaryoblastic leukemia (AMKL) is a subtype of poor prognosis acute myeloid leukemia (AML) affecting primarily young children. Recently, the ETO2-GLIS2 fusion has been identified in 20-30% of de novo AMKL and associated with the worst prognosis in this subtype of AML. To characterize the transformation induced by ETO2-GLIS2, we first defined the consequences of ETO2-GLIS2 expression on hematopoietic progenitors and the contribution of ETO2 and GLIS2 on differentiation and self-renewal. Using methylcellulose replating assays and phenotype characterization, we show that the GLIS2 moiety drives the megakaryocytic phenotype whereas both the ETO2 and GLIS2 moieties are required for maintaining self-renewal. Global expression profiling and comparison to patients' signature consistently identify ETO2-GLIS2-mediated deregulation of major transcriptional regulators of hematopoiesis and leukemogenesis, including overexpression of the ERG oncogene. ChIP-seq analysis reveals that ETO2-GLIS2 is recruited at normal ETO2 complexes sites and also at GLIS2-specific targets through binding via GLIS2 DNA-binding domain. We demonstrate that ETO2-GLIS2 fusion localize at half of H3K27Ac-dense enhancers, so called super-enhancers, to control transcription of associated genes. We show that interaction of ETO2-GLIS2 with ETO2 complexes is an essential node for the transcriptional control by the fusion at enhancer elements. Indeed, ETO2-GLIS2 dimerizes and interacts with endogenous ETO2 via its NHR2 domains. An NHR2 peptide-interference strategy inhibits oligomerization, reverses the transcriptional activation at enhancers, promotes megakaryocytic differentiation and abrogates human AMKL cells maintenance in vivo. Finally, upregulation of ERG by ETO2-GLIS2 further strengthen enhancers formation as ERG is co-recruited generating a feed forward loop at these elements and its knockdown or genetic inactivation downregulates expression of ETO2-GLIS2 targets required for leukemic cells survival. We propose that the megakaryocytic differentiation arrest and self-renewal controlled by ETO2-GLIS2 results from an imbalance in the expression of master transcription factors imposed by aberrant chromatin structures at enhancers that may be disrupted by targeting the NHR2 interface. Disclosures No relevant conflicts of interest to declare.

Background: This year, germline predisposition to haematological malignancy (HM) debuts in the World Health Organization classification of myeloid neoplasms and acute leukemia (Blood, 2016;127:2391). It has been 17 years since germline mutations in RUNX1 were found to lead to familial platelet disorder (FPD) with predisposition to myelodysplastic syndrome and acute myeloid leukaemia (MDS/AML) (Nat Genet. 1999;23:166). Now, nearly 80 families have been reported with damaging germline mutations or deletions affecting RUNX1 function, associated with FPD, making it an increasingly significant clinical presence. Although thrombocytopenia and platelet dysfunction are present in almost all RUNX1 mutant carriers, we and others have observed that the predisposition to HM varies between family members, with respect to age at diagnosis and the type of malignancy, and in some cases RUNX1 mutation carriers have no apparent HM development over their lifespan. The reasons for this heterogeneity are currently unknown. Aims: We are conducting an international collaborative study examining RUNX1 mutated families. The aim of the research project is to classify the range of phenotypes correlated with RUNX1 mutations comprehensively (including non-malignant phenotypes such as skin disorders) and to determine if the type of RUNX1 mutation and the presence of other germline and acquired mutations in relevant HM genes correlate with the likelihood of HM development, or the type of HM that develops. Across all of our data we aim to analyse clinically relevant information that will be used to inform prognosis and clinical management in germline RUNX1 mutation carriers. Results:From a review of the literature for previously characterised RUNX1 mutant families most mutations are predicted to be loss-of-function, with the combination of frameshift, stopgain, splicing and deletion accounting for the majority of alterations (57, 70%) compared to missense mutations (22, Figure 1). The most common sites of mutation are R201 and R204, affected by both missense and stopgain (10 total), which lie within the nuclear localisation signal at the end of the RUNT domain (Figure 1). We also surveyed in detail 12 RUNX1 pedigrees with both novel and previously described missense, frameshift, stopgain and deletion mutations and found that, while all families developed myeloid malignancies, 6 families also had individuals who developed lymphoid malignancy (most often Acute lymphoblastic leukemia (ALL)) which was heritable in sub-families, and subject to anticipation (e.g see IV-5 and V-5 in Figure 2). Consistent with population genome wide association studies identifying RUNX1 as a susceptibility locus for psoriasis (J Autoimmun. 2015;64:66), we find that skin conditions (psoriasis, eczema) are common, and present in germline RUNX1 carriers in 50% of our families; most commonly observed in families with stopgain and frameshift mutations. Genomic analysis of selected samples confirms that mutation of the other RUNX1 allele is the most commonly acquired mutation in germline RUNX1 mutation carriers developing HM. Alterations of chromosomes 21 and 7 are also common. DNMT3A and PHF6 acquired mutations were the next most frequently observed in tumors and mutations in U2AF1 and ASXL1 in the blood of RUNX1 carriers without HM were observed, suggestive of pre-HM clonal expansion. Finally, in a family with a novel R169I RUNX1 mutation, a rare germline ASXL1 variant (E1102D, 1.0% in ExAC) was found in two RUNX1 carriers who developed early onset AML. This variant is also significantly enriched in an MDS cohort unselected for family history compared to the general population (HR 1.3, p=0.02), as well as ASXL1 N986S (0.1% in ExAC, HR 3.3, p=0.0002) suggesting they operate as germline HM risk modifiers. Interestingly RUNX1 and ASXL1 acquired mutations often co-occur in sporadic MDS/AML and our data suggests this collaboration may also occur at the germline level. Conclusions:Annotation of skin phenotypes co-existent with a family history of haematological malignancy may assist in identifying RUNX1 mutant families. Both acquired and germline mutations in known HM genes may modify germline RUNX1 driven HM penetrance and phenotype. Our data suggest that screening of RUNX1 germline mutation carriers for germline and acquired variants in other HM genes could provide an important tool for defining risk and requires further investigation. Disclosures Owen: Pharmacyclics: Research Funding; Janssen: Honoraria; Roche: Honoraria, Research Funding; Novartis: Honoraria; Gilead: Honoraria, Research Funding; Lundbeck: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Abbvie: Honoraria. Godley:UpToDate: Honoraria; Onconova, Inc.: Research Funding.

The transcription factor encoded by the E-twenty-six (ETS)-related gene, ERG , is an essential regulator of hematopoietic stem cell function and a potent human oncoprotein. Enforced expression of ERG in murine hematopoietic cells leads to the development of a well-characterized lymphoid leukemia and a less well-defined non lymphoid disease. To clarify the latter, we generated murine bone marrow chimeras with enforced Erg expression in engrafted hematopoietic progenitor cells. As expected, these mice developed lymphoid leukemia. However, the previously reported non lymphoid disease that developed was shown to be a uniform, transplantable leukemia with both erythroid and megakaryocytic characteristics. In vivo, this disease had the overall appearance of an erythroleukemia, with an accumulation of immature erythroblasts that infiltrated the bone marrow, spleen, liver, and lung. However, when stimulated in vitro, leukemic cell clones exhibited both erythroid and megakaryocytic differentiation, suggesting that transformation occurred in a bipotential progenitor. Thus, in mice, Erg overexpression induces the development of not only lymphoid leukemia but also erythro-megakaryocytic leukemia.

Novel RUNX1 mutations in familial platelet disorder with enhanced risk for acute myeloid leukemia: clues for improved identification of the FPD/AML syndrome

Down syndrome (DS) persons are born with various hematopoietic abnormalities, ranging from relatively benign, such as neutrophilia and macrocytosis, to a more severe transient myeloproliferative disorder (TMD). In most cases, these abnormalities resolve in the first few months to years of life. However, sometimes the TMD represents a premalignant disease that develops into acute megakaryocytic leukemia (AMKL), usually in association with acquired GATA1 mutations. To gain insight into the mechanisms responsible for these abnormalities, we analyzed the hematopoietic development of the Ts1Cje mouse model of DS. Our analyses identified defects in mature blood cells, including macrocytosis and anemia, as well as abnormalities in fetal liver and bone marrow stem and progenitor cell function. Despite these defects, the Ts1Cje mice do not develop disease resembling either TMD orAMKL, and this was not altered by a loss of function allele of Gata1. Thus, loss of Gata1 and partial trisomy of chromosome 21 orthologs, when combined, do not appear to be sufficient to induce TMD or AMKL-like phenotypes in mice usc Refereed/Peer-reviewed

The Ts65Dn mouse is the most widely investigated segmentally trisomic mouse model of Down syndrome. Quantitative PCR based methods are the preferred way of detecting the trisomic segment for genotyping purposes. However, identification of a 1.5 fold difference in target DNA is at the limit of detection of most quantitative PCR based methods, and in practice this can lead to difficulties in assigning genotypes. We report a 100% accurate multiplex ligation-dependent probe amplification (MLPA) assay for genotyping the Ts65Dn mouse that is also applicable to all other segmentally trisomic mouse models of Down syndrome.

Sampling and analyzing new families with inherited blood disorders are major steps contributing to the identification of gene(s) responsible for normal and pathologic hematopoiesis. Familial occurrences of hematological disorders alone, or as part of a syndromic disease, have been reported, and for some the underlying genetic mutation has been identified. Here we describe a new autosomal dominant inherited phenotype of thrombocytopenia and red cell macrocytosis in a four-generation pedigree. Interestingly, in the youngest generation, a 2-year-old boy presenting with these familial features has developed acute lymphoblastic leukemia characterized by a t(12;21) translocation. Tri-lineage involvement of platelets, red cells and white cells may suggest a genetic defect in an early multiliear progenitor or a stem cell. Functional assays in EBV-transformed cell lines revealed a defect in cell proliferation and tubulin dynamics. Two candidate genes, RUNX1 and FOG1, were sequenced but no pathogenic mutation was found. Identification of the underlying genetic defect(s) in this family may help in understanding the complex process of hematopoiesis.

Analyses of chromosomal translocation and inversion breakpoints in sporadic acute myeloid leukemias have identified many transcription factors as playing a role in leukemogenesis. Studies of families with a Mendelian predisposition to hematological malignancies have identified the gene coding for the transcription factor RUNX1 as a leukemia-predisposing gene involved in the first steps of leukemogenesis. Using two families, another autosomal dominant familial leukemia locus was linked to chromosome band 16q22 where the CBFB gene maps. Although CBFB forms a core-binding factor transcriptional complex with RUNX1, previous analyses have excluded the CBFB gene as the leukemia-predisposing gene in these families. In the current study, we performed an extended molecular analysis in these families of the four other transcription factor genes in the 16q22 critical region as well as of two other genes with a known association with leukemia. Several previously undescribed but nonpathogenic sequence variants were identified. We demonstrated that the transcription factors E2F4, CTCF, NFATC3, and NFAT5, and the genes coding for NAD(P)H:quinone oxido-reductase 1 (NQO1) and for E-cadherin are not responsible for the leukemia susceptibility in these families. The presence of NQO1 polymorphisms may suggest a role for this gene in disease risk modification in these families. © 2004 Wiley-Liss, Inc.

Krajem 18. stoljeća njemačka je etnografska literatura ustanovila da među narodima postoje kulturne razlike, a varijante kao što su jezik ili religija ustanovljene su genetskim, a ne okolišnim čimbenicima. U istoj se literaturi susreću termini Volksmoral, koji podrazumijeva utemeljene kriterije o urednosti naroda, njegovoj radinosti, te političkoj tradiciji, i Volksgeist, prirodan nacionalni duh koji je zaslužan za osnovna obilježja naroda i na temelju kojega se baziraju patriotizam i nacionalni ponos. Zanimanje za porijeklo naroda krajem 18. stoljeća bilo je usko povezano s razvojem nacionalne svijesti. U to je doba Johann Gottfried von Herder, teoretičar kulturnog nacionalizma, svojim radom uvelike utjecao na znanstvenike srednje i istočne Europe i tako utro put kasnijem političkom nacionalizmu. Prema M. Burleighu i W. Wippermanu, Herderov je kulturni nacionalizam iskorišten za legitimizaciju njemačke vladavine nad bivšim zapadno-slavenskim i poljskim područjima. Područja istočne i srednje Europe sa stoljetnom etničkom i lingvističkom raznolikošću (Silezija, Transilvanija, Habsburški Jadran), postala su pogodno područje za ispitivanje (ili potvrđivanje) takvih teorija o etnicitetu. U čitavom su nizu tekstova istaknuti izrazi kojima putopisci kao predstavnici "više" kulture ocjenjuju druge etničke skupine, uglavnom tretirane kao primjer inferiornih kultura, stvarajući pritom relativno ograničen niz stereotipa. Posebna se pozornost posvećuje tekstovima o austrijskoj Istri nastalima između 1770. i 1815. godine. Stereotipi o stanovnicima te regije fiksirani su i klišeizirani, posve analogno onima koji nastaju o stanovnicima drugih slavenskih zemalja. Istra se smatra jugom, područjem na kojem se govori mješavina različitih jezika upitnoga statusa, sa stanovništvom uglavnom negativnih moralnih osobina. U njemački se pisanim tekstovima osjeća aroma kolonijalnog diskursa kad slavenskog seljaka svode na prirodno biće kojemu je omogućeno uživanje u jednostavnosti života zbog svoje neopterećenosti civilizacijskim pogodnostima i napretkom. Takva percepcija istarskog čovjeka i prostora utjecala je na političke programe svih etničkih skupina koje su na njemu živjele, naime podjela Istre je bila anticipirana kad su Europljani počeli graditi svoje identitete u skladu s percipiranim etničkim, a ne regionalnim ili patriotskim razdvojnicama. Drugim riječima, nacionalizmi koji su se u Istri pojavili kasnije, u 19. stoljeću, barem su dijelom bili proizvod određenog trenutka u raščlambi i opisu europskih kultura, obilježenoga rasističkim diskursom., The ethnological writings from the late eighteenth century noticed that there were cultural differences among peoples. Those differences were attributed to genetic factors and to innate national spirit of each nation, both of which are responsible for a nation's basic characteristics. The paper deals primarily with the reports on Austrian Istria, written between 1770 and 1815, in which stereotypes about the population are fixed and turned into clichés the same way as about the population of other Slavic countries. This perception of Istria affected the political programmes of all ethnic groups that lived there, as well as the later division of Istria.

Abstract 1283 ETS-related gene, ERG, is a key regulator of hematopoietic stem cell (HSC) function, and a potent oncogene. It is involved in chromosomal translocations with the EWS gene in Ewing's sarcoma, the TLS gene in Acute Myeloid Leukemia (AML) and the TMPRSS2 gene in more than half of all prostate cancers. In addition, increased ERG levels are associated with poor prognosis in cytogenetically normal AML and T-cell Acute Lymphoblastic Leukemia; and our recent data suggests that trisomy of ERG is important for development of myeloproliferative disease and Acute Megakaryocytic Leukemia (AMKL) in Down syndrome individuals. The role ERG, and the oncogenic fusion protein TLS-ERG, play during hematopoietic transformation remains unclear. Hematopoietic overexpression of ERG has been shown to induce T-cell leukemia in mice. Development of a non-lymphoid disease has also been described, however this disease was reported to be an AMKL by one group, and a non-malignant erythroid hyperplasia by another. Hematopoietic overexpression of TLS-ERG in mice has not been described. This fusion has been shown to perturb differentiation and increase self-renewal of human myeloid progenitor cells in vitro, and enable the IL-3 dependent L-G murine myeloid progenitor cell line to induce a leukemia-like disease in vivo. In order to clarify and compare the role of wild-type and rearranged forms of ERG in leukemia development, we injected lethally irradiated mice with fetal liver cells (FLCs) transduced with retrovirus carrying either Erg or TLS-ERG. These mice succumbed to disease with a median latency of 80 days after receiving Erg-transduced FLCs, or 44 days after receiving TLS-ERG-transduced FLCs. Consistent with published data, 30% of Erg mice developed T-cell leukemias. Interestingly, no TLS-ERG mice developed this disease. Strikingly, 100% of Erg and TLS-ERG mice developed an identical non-lymphoid disease characterised by hepatosplenomegaly, anemia and leukocytosis. Histopathological and flow cytometric analysis revealed infiltration of the bone marrow, spleen, lung and liver by nucleated erythroblasts, expressing a high level of CD71 and varying levels of Ter119. Interestingly, in some mice a subset of these cells also expressed the megakaryocytic marker CD41. Primary spleen cells were capable of transplanting disease in non-irradiated mice, demonstrating that this disease was malignant. Spleen cells from Erg and TLS-ERG leukemic mice, but not controls, were capable of generating large numbers of small colonies when cultured in methylcellulose stimulated with IL-3/SCF/EPO. These colonies primarily contained erythroblasts (CD71+Ter119+/−), however some cells also expressed CD41 and were acetylcholinesterase positive. Most acetylcholinesterase positive cells were of small size, indicating either incomplete or early megakaryocyte maturation. Combined, these data suggest that hematopoietic overexpression of Erg or TLS-ERG in mice leads to transformation of a bi-potential erythroid-megakaryocyte progenitor. Following transformation, this cell appears to retain some bi-potentiality in vitro, however in vivo it primarily develops along the erythroid lineage. Thus, Erg- and TLS-ERG- induced non-lymphoid disease may be best described as an erythro-megakaryocytic leukemia. Finally, the data also indicate that truncation and fusion to TLS abrogates ERG's ability to transform lymphoid progenitors, however TLS-ERG retains the ability to transform myeloid progenitors in a manner that strongly resembles that of wild-type Erg. Disclosures: No relevant conflicts of interest to declare.

Abstract LBA-3 Mutations in the transcription factor genes, RUNX1 and CEBPA, can lead to an autosomal dominant familial predisposition to MDS/AML. Using a candidate gene approach, we have detected domain specific heterozygous mutations in the GATA2 gene in 4 MDS/AML families which predispose to MDS/AML. The same novel heterozygous T354M missense mutation was observed in 3 families and a 355delT mutation in 1 family, all with multigenerational transmission of MDS and/or MDS/AML. Importantly, these genetic variants segregate with all affected members in each of the families. The 2 mutated threonine residues are in 5 consecutive highly conserved threonine residues at the DNA-binding, protein-protein interacting second zinc finger (ZF2) of GATA2. Neither these mutations, nor any other variants in the GATA2 coding sequence, were seen in a population screen of 695 normal individuals. Haplotype analysis suggests that the T354M mutation has multiple ancestral origins. While mutations in RUNX1 and CEBPA, can also lead to familial predisposition to MDS/AML, these patients with GATA2 mutations are unique in that there is no obvious pre-MDS or pre-leukaemic phenotype such as thrombocytopenia (RUNX1) and eosinophilia (CEBPA) in predisposed carriers. Most patients in these families have had a rapid disease course “appearing out of the blue” leading to death, with a variety of ages of onset from teenagers to early 40s. Yet remarkably, there are still asymptomatic carriers in their 60s. One of these carriers, and his 2 children, has had bone marrow prophylactically stored over 15 years ago in case of disease onset. No pathogenic GATA2 coding sequence changes were found in 268 sporadic MDS/AML patient samples. Additionally, GATA2 mutations were not found in germline samples from 35 other families predisposed to AML and various other hematological malignancies. Both the T354M and 355delT mutants appear to localize appropriately to the nucleus and maintain at least some DNA binding in electrophoretic mobility shift assays. We used the known murine Gata3 ZF2 structure bound to DNA to model the effects of the observed mutations and demonstrated that the T354 residue does not contact DNA but makes polar contact with the adjacent threonines, and via its amino group, with C349 which coordinates the zinc atom. Replacement of the T354 side-chain with the bulky methionine moiety may affect the zinc contacts and is predicted to alter the overall structure of this ZF2. In contrast, 355delT will shorten the conserved threonine string which is predicted to impact on the orientation and position of L359 which directly contacts DNA. Thus, 355delT is likely to have an effect on DNA binding. Luciferase reporter assays indicate that T354M and 355delT greatly reduce the transactivation ability of GATA2 on multiple response elements, impacting on downstream target genes such as RUNX1 and CD34. Of note, T354M shows a markedly lesser synergistic effect than wildtype (WT) GATA2 with PU.1 on the CSF1R promoter. Competition assays show that these mutations may be acting in a dominant negative fashion in some biological contexts. In stable promyelocytic HL-60 cell lines expressing regulatable GATA2 (WT or T354M), T354M allows proliferation to proceed even under stimulus to differentiate with all-trans retinoic acid. Microarray studies indicated that the down regulation of proapoptotic BCL-xS by T354M, but not WT, may be responsible for this phenotype. GATA2 is considered to be a hematopoietic “stemness” gene, highly expressed in haematopoietic stem cells and is required for megakaryocyte and mast cell production. GATA2 is down regulated during myeloid differentiation and forced overexpression prevents such differentiation. Discovery of GATA2 mutants in MDS/AML predisposed families provides new tools for probing the mechanism of GATA2 induced leukemogenesis, and possibly also for clarifying its role in maintenance of stemness. Our findings highlight the power of investigating familial predispositions to cancer identifying specific mutations with unique biological effects. They have immediate implications for diagnostic genetic testing, and longer term therapeutic implications through identification of drugable biological pathways such as apoptosis. The poor outcome associated with these mutations may suggest that an aggressive strategy is appropriate in the treatment of affected individuals in families found to be carrying GATA2 mutations. Disclosures: No relevant conflicts of interest to declare.

Aim To identify the causative heritable mutation in a family with autosomal dominant familial platelet disorder with predisposition to acute myeloid leukemia (FPD/AML). Method Confirmation of family pedigree, enrolment into ethics committee approved Australian Familial Hematological Cancer Study, procurement of genomic DNA from pedigree members and genetic analysis by sequencing of RUNX1 and CEBPA genes. Results The proband intially presented aged 50 with mild thrombocytopenia initially diagnosed as idiopathic thrombocytopenic purpura when bone marrow examination (including cytogenetics analysis) was normal. Three years later she was referred with severe progressive thrombocytopenia unresponsive to high dose steroids and intravenous immunoglobulin together with mild anemia and neutropenia. Marrow examination revealed subtle dysplasia with monosomy 7 in 12/20 metaphases. A diagnosis of myelodysplastic syndrome (MDS) was made. Three months later (Feb 2007), she progressed to acute myeloid leukemia (AML). The proband’s mother had had mild thrombocytopenia with subsequent MDS (aged 70) and died of AML two years later. The proband’s only sibling and nephew have mild thrombocytopenia without features of MDS. The proband entered cytogenetic remission following one course of AML induction therapy but continued to show dysplastic features. She then received 2 cycles of consolidation therapy and has undergone unrelated donor allogeneic stem cell transplant (July 2007). Sequencing of PCR products from exons of the RUNX1 gene identified a novel heterozygous mutation in the proband’s constitutional DNA: c.958C>T in exon 7, in the transactivation domain, causing a nonsense mutation, p.Arg293X (sequence variation for RUNX1 classified according to GenBank Accession No. NC_000021). The sibling has the same mutation and studies in other relatives are underway. Conclusion This study has identified a novel RUNX1 mutation responsible for FPD/AML in this family. Clinicians should be aware of this rare inherited disorder and the availability of genetic testing. People with mutations may be asymptomatic and genetic testing in such families is essential before considering bone marrow transplantation from a living related donor.

BackgroundTheRUNX1transcription factor gene is frequently mutated in sporadic myeloid and lymphoid leukemia through translocation, point mutation or amplification. It is also responsible for a familial platelet disorder with predisposition to acute myeloid leukemia (FPD-AML). The disruption of the largely unknown biological pathways controlled by RUNX1 is likely to be responsible for the development of leukemia. We have used multiple microarray platforms and bioinformatic techniques to help identify these biological pathways to aid in the understanding of why RUNX1 mutations lead to leukemia.ResultsHere we report genes regulated either directly or indirectly by RUNX1 based on the study of gene expression profiles generated from 3 different human and mouse platforms. The platforms used were global gene expression profiling of: 1) cell lines with RUNX1 mutations from FPD-AML patients, 2) over-expression of RUNX1 and CBFβ, and 3) Runx1 knockout mouse embryos using either cDNA or Affymetrix microarrays. We observe that our datasets (lists of differentially expressed genes) significantly correlate with published microarray data from sporadic AML patients with mutations in eitherRUNX1or its cofactor,CBFβ. A number of biological processes were identified among the differentially expressed genes and functional assays suggest that heterozygousRUNX1point mutations in patients with FPD-AML impair cell proliferation, microtubule dynamics and possibly genetic stability. In addition, analysis of the regulatory regions of the differentially expressed genes has for the first time systematically identified numerous potential novel RUNX1 target genes.ConclusionThis work is the first large-scale study attempting to identify the genetic networks regulated by RUNX1, a master regulator in the development of the hematopoietic system and leukemia. The biological pathways and target genes controlled by RUNX1 will have considerable importance in disease progression in both familial and sporadic leukemia as well as therapeutic implications.