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Ubiquitin-specific peptidase 15 (USP15) is a deubiquitinating enzyme implicated in critical cellular and oncogenic processes. We report that USP15 mRNA and protein are overexpressed in human acute myeloid leukemia (AML) as compared to normal hematopoietic progenitor cells. This high expression of USP15 in AML correlates with KEAP1 protein and suppression of NRF2. Knockdown or deletion of USP15 in human and mouse AML models significantly impairs leukemic progenitor function and viability and de-represses an antioxidant response through the KEAP1-NRF2 axis. Inhibition of USP15 and subsequent activation of NRF2 leads to redox perturbations in AML cells, coincident with impaired leukemic cell function. In contrast, USP15 is dispensable for human and mouse normal hematopoietic cells in vitro and in vivo. A preclinical small-molecule inhibitor of USP15 induced the KEAP1-NRF2 axis and impaired AML cell function, suggesting that targeting USP15 catalytic function can suppress AML. Based on these findings, we report that USP15 drives AML cell function, in part, by suppressing a critical oxidative stress sensor mechanism and permitting an aberrant redox state. Furthermore, we postulate that inhibition of USP15 activity with small molecule inhibitors will selectively impair leukemic progenitor cells by re-engaging homeostatic redox responses while sparing normal hematopoiesis.

Germline mutations in the RNA Helicase gene DDX41 are the most common cause of inherited susceptibility to adult MDS and AML. These mutations are always heterozygous and are typically frameshifts, causing loss of functional protein. We recently reported that at least one functional copy of DDX41 is essential for hematopoiesis, and that DDX41 is required for ribosome biogenesis (Chlon et al., Cell Stem Cell 2021). While biallelic DDX41 mutations cause dramatic defects in hematopoiesis, the role of heterozygous mutations in MDS pathogenesis is not yet understood. DDX41 mutation carriers frequently experience idiopathic cytopenias of unknown significance (ICUS) prior to MDS onset, suggesting that underlying hematopoietic defects contribute to MDS/AML (Choi et al., Haemotologica 2021). The majority of DDX41-mutant MDS patients have refractory anemia, indicating that the erythroid lineage is particularly affected in these patients (Sebert et al., Blood 2019). Since ribosome defects are a common cause of inherited anemias and also contribute to MDS pathogenesis, we characterized the effect of heterozygous DDX41 mutations on erythropoiesis in murine and human models. Mice that were transplanted with Ddx41+/− bone marrow develop anemia at 12-15 months post-transplant. At younger ages, these mice have normal hematopoietic indices, but when stress erythropoiesis is induced by Pheylhydrazine-treatment, the Ddx41+/− mice have prolonged anemia. These observations indicate that heterozygous loss of DDX41 causes defects in erythropoiesis during stress and aging. We further characterized the effect of Ddx41-hetrozyogisty on erythroid progenitor function in vitro. In colony assays, we found that Ddx41+/− HSPC form fewer BFU-E but comparable numbers of myeloid colonies. In liquid culture erythroid differentiation cultures, we found that Ddx41+/− HSPC produce fewer CD71+ Ter119+ progenitors than controls. Mechanistically, we found that in vitro-derived erythroid progenitors from both mice and cell line models had decreased protein translation, suggesting that ribosome defects underlie the observed inefficiency in erythropoiesis. In congenital ribosomopathy diseases, ribosome defects lead to p53 activation which contributes to defects in erythropoiesis. Interestingly, TP53 mutations are common in DDX41-mutant MDS/AML (Sebert et al., Blood 2019), suggesting that the ribosome deficiency selects for these mutations. To assess the role of p53 mutations in the development of DDX41-mutant MDS, we generated Ddx41+/−;p53+/− mice and transplanted the bone marrow into lethally-irradiated recipients and followed the mice for 1 year. These mice developed a lethal MDS-like phenotype at 7-12 months post-transplant while control mice lived until the end of the study period. The sick mice had anemia and other cytopenias accompanied by enlarged spleens and dysplastic myeloid cells. Collectively, these results indicate that p53 mutations cooperate with Ddx41-heterozygosity to promote hematologic malignancy. Citation Format: Timothy M Chlon, Emily Stepanchick, Analise Sulentic, Andrew Wilson, Daniel T Starczynowski. Heterozygous mutations in DDX41 cause erythroid progenitor cell defects and cooperate with p53 mutations to cause hematologic malignancy [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A46.

DDX41 mutations are the most common germline alterations in adult myelodysplastic syndromes (MDSs). The majority of affected individuals harbor germline monoallelic frameshift DDX41 mutations and subsequently acquire somatic mutations in their other DDX41 allele, typically missense R525H. Hematopoietic progenitor cells (HPCs) with biallelic frameshift and R525H mutations undergo cell cycle arrest and apoptosis, causing bone marrow failure in mice. Mechanistically, DDX41 is essential for small nucleolar RNA (snoRNA) processing, ribosome assembly, and protein synthesis. Although monoallelic DDX41 mutations do not affect hematopoiesis in young mice, a subset of aged mice develops features of MDS. Biallelic mutations in DDX41 are observed at a low frequency in non-dominant hematopoietic stem cell clones in bone marrow (BM) from individuals with MDS. Mice chimeric for monoallelic DDX41 mutant BM cells and a minor population of biallelic mutant BM cells develop hematopoietic defects at a younger age, suggesting that biallelic DDX41 mutant cells are disease modifying in the context of monoallelic DDX41 mutant BM.

Chronic innate immune signaling in hematopoietic cells is widely described in myelodysplastic syndromes (MDS), and innate immune pathway activation, predominantly via pattern recognition receptors, increases the risk of developing MDS. An inflammatory component to MDS has been reported for many years, but only recently has evidence supported a more direct role of chronic innate immune signaling and associated inflammatory pathways in the pathogenesis of MDS. Here we review recent findings and discuss relevant questions related to chronic immune response dysregulation in MDS.

Germline mutations in the RNA Helicase gene DDX41 cause inherited susceptibility to Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukemia (AML). These mutations are always heterozygous and are typically frameshifts, causing loss of protein expression. We recently reported that at least one functional copy of DDX41 is essential for hematopoiesis, and that DDX41 is required for ribosome biogenesis. While biallelic DDX41 mutations cause dramatic defects in hematopoiesis, the role of heterozygous mutations in Myelodysplastic Syndrome pathogenesis is not yet understood. Recent clinical studies have pointed out that some patients bearing germline DDX41 mutations have idiopathic cytopenias of unknown significance (ICUS) prior to MDS onset, suggesting that underlying hematopoietic defects precede and potentially contribute to the onset of MDS/AML (Choi et al., Haemotologica 2021). It has also been noted that the majority of DDX41-mutant MDS patients have refractory anemia, indicating that the erythroid lineage is particularly effected in these patients (Sebert et al., Blood 2019). Since ribosome defects are a common cause of inherited anemias and also contribute to MDS pathogenesis, we characterized the effect of heterozygous DDX41 mutations on erythropoiesis in murine and human models. Mice that have been transplanted with Ddx41 +/- bone marrow develop anemia at 12-15 months post-transplant, indicating that detection of erythroid defects in vivo is aging-dependent. We characterized the effect of heterozygosity of Ddx41 on erythroid progenitor function in vitro and found that Ddx41 +/- bone marrow from young mice yields fewer BFU-E in colony assays but comparable numbers of myeloid colonies. Liquid culture erythroid differentiation of Ddx41 +/- bone marrow produces fewer CD71+ Ter119+ progenitors than controls. To characterize the effect of heterozygous DDX41 mutations on human erythropoiesis, we generated induced pluripotent stem cells bearing heterozygous frameshift mutations in DDX41 using CRISPR. We found that these DDX41 +/- iPSC lines produced CD43+/CD34+ hematopoietic progenitor cells (HPC) with equal efficiency as unmodified control iPSC. However, once these HPC were induced to differentiate down the erythroid lineage in liquid culture, they made fewer CD71+ GLYA+ erythroid progenitors and fewer hemoglobinized cells. The DDX41 +/- HPC also produced fewer BFU-E in colony assays. Mechanistically, we found that the in vitro-derived erythroid progenitors from both mice and human iPSC had decreased protein translation, suggesting that ribosome defects underlie the observed erythroid differentiation defects. In diseases such as Diamond Blackfan Anemia and Dyskeratosis Congenita, ribosome defects lead to p53 activation which reduces cell cycle progression in erythroid progenitors. To test the role of p53 in the erythroid defects caused by Ddx41 heterozygosity, we crossed Ddx41 +/- mice with p53-knockout mice and found that loss of p53 fully rescued the BFU-E colony formation of Ddx41 +/- bone marrow HPC. We confirmed this finding using CRISPR-mediated knockout of p53 in Ddx41 +/- BM HPC. Collectively, these results suggest that a mild ribosome defect in DDX41 +/- HPC causes a deficit in erythropoiesis that results in anemia with aging. It is likely that this anemia causes stress in the bone marrow and a selective environment in which malignant hematopoietic stem and progenitor cells arise, leading to MDS and AML. Disclosures Starczynowski: kurome Inc: Consultancy.

Squamous cell carcinoma (SCC) is a global public health burden originating in epidermal stem and progenitor cells (ESPCs) of the skin and mucosa. To understand how genetic risk factors contribute to SCC, studies of ESPC biology are imperative. Children with Fanconi anemia (FA) are a paradigm for extreme SCC susceptibility caused by germline loss-of-function mutations in FA DNA repair pathway genes. To discover epidermal vulnerabilities, patient-derived pluripotent stem cells (PSCs) conditional for the FA pathway were differentiated into ESPCs and PSC-derived epidermal organotypic rafts (PSC-EORs). FA PSC-EORs harbored diminished cell-cell junctions and increased proliferation in the basal cell compartment. Furthermore, desmosome and hemidesmosome defects were identified in the skin of FA patients, and these translated into accelerated blistering following mechanically induced stress. Together, we demonstrate that a critical DNA repair pathway maintains the structure and function of human skin and provide 3D epidermal models wherein SCC prevention can now be explored.

Germline mutations in the DEAD-box RNA helicase gene DDX41 cause inherited susceptibility to Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukemia (AML). These patients have normal hematopoietic indices into adulthood and present with MDS at a median age of 61 years, slightly younger than the general MDS patient population (71 years). Germline DDX41 mutations are always heterozygous and are typically frame-shift mutations, causing loss of function of the protein. More than half of these patients acquire a second-hit mutation in the healthy DDX41 allele in their disease clones. Greater than 80% of the second-hit mutations cause the amino acid substitution R525H, which results in loss of helicase activity. Multiple functions have been ascribed to DDX41, such as functioning as an innate immune sensor, a component of the RNA spliceosome, and a regulator of ribosomal RNA processing; however, its role in the pathogenesis of MDS remains poorly understood. To mimic the DDX41 mutations in human patients, we generated conditional mouse models of the most common DDX41 mutations, D140fs and R525H. We found that DDX41 is essential for the viability and function of hematopoietic stem and progenitor cells (HSPCs) and that R525H mutations render DDX41 inactive. We have also reported that mice with biallelic DDX41 mutations develop progressive anemia, dysplasia, and marrow failure. HSPCs lacking wild-type Ddx41 have dysfunctional ribosomes and reduced protein translation, leading to cycle arrest and apoptosis. As a mechanistic basis for the ribosome defect in Ddx41-deficient HSPCs, we found that loss of Ddx41 causes a profound increase in unprocessed snoRNA transcripts, which likely interrupts their cellular function. Importantly, we did not observe a change in host gene transcript abundance, and thus it is unlikely that the defects observed in Ddx41-deficient cells are caused by loss of the protein products of the host genes. SnoRNAs regulate ribosomal RNA (rRNA) by catalyzing post-transcriptional modifications, including methylation and pseudouridylation. Since the majority of the snoRNAs that were found to be unprocessed in Ddx41-deficient HSPCs were from the SNORA family, which is involved in pseudouridylation, we quantified the abundance of pseudouridine at specific sites in ribosomal RNA and found it to be reduced in Ddx41-deficient HSPCs. Utilizing global high-throughput sequencing approaches to analyze DDX41 binding to RNAs, we found that DDX41 preferentially binds to snoRNAs relative to all other types of RNA in the cells. Thus, DDX41 binds to snoRNAs and is essential for snoRNA processing, snoRNA-mediated rRNA pseudouridylation, and protein translation in HSPCs. These findings provide critical mechanistic insight into the protein translation defect that we and others have observed in DDX41-deficient cells and uncover the basis of ineffective hematopoiesis in MDS patients with DDX41 mutations. Disclosures Starczynowski: Tolero Therapeutics: Research Funding; Kurome Therapeutics: Consultancy, Current equity holder in private company, Research Funding; Captor Therapeutics: Consultancy.

Myelodysplastic syndromes (MDS) are hematopoietic stem cell (HSC) disorders in which myeloid cell differentiation is impaired, causing blood lineage cytopenias and potentially leading to acute myeloid leukemia (AML) through malignant transformation. MDS occurs in adults with a median age of 71 years, and is associated with multiple cytogenetic and genetic abnormalities in the diseased HSC. Younger patients can also have MDS as a result of underlying congenital diseases or secondary effects from cancer therapy. It has recently been discovered that some families with high rates of MDS incidence bear heterozygous inherited mutations in DDX41, a member of the DEAD box RNA helicase family of genes. These patients typically have normal hematopoietic indices into adulthood and present with MDS at a median age of 61 years, slightly younger than the general population. Inherited DDX41 mutations are always heterozygous and are typically frame-shift mutations, indicating they are likely loss of function. Approximately half of MDS patients with inherited DDX41 mutations acquire a second-hit, often R525H, in the healthy DDX41 allele in their disease clones. This mutation is also observed in 1-2% of de novo AML patients, suggesting it causes gain of function or dominant negative activity. Multiple functions have been ascribed to DDX41, such as functioning as an innate immune sensor and as an RNA splicing regulator, but its role in the pathogenesis of MDS remains unknown. We set out to model DDX41 mutations by generating conditional DDX41 knockout and R525H-knock-in mice. Combining these alleles and crossing to Rosa-Cre-ERT expressing mice allowed for tamoxifen-inducible acquisition of knockout (KO), heterozygous (HET), heterozygous knock-in (KI/+) and knock-in alone (KI/-) HSPC. The KO and KI/- HSPC were incapable of engrafting into recipient mice and underwent rapid cell cycle arrest and apoptosis, indicating that Ddx41 is required for HSPC cell viability and that the R525H mutation causes loss of the required function. In contrast, the HET and KI/+ HSPC survived and proliferated normally in culture and successfully engrafted irradiated recipient mouse bone marrow. HET and KI/+ transplanted mice had increased numbers of LSK cells, and subset of mice developed a myeloid malignancy, resembling the human disease. To determine the function of DDX41 that is critical for hematopoiesis, we performed a tandem-pulldown followed by mass spectrometry analysis to identify relevant DDX41 interacting proteins in human AML cells. We found that DDX41 interacts with multiple proteins in the small ribosomal subunit, including RPS3 and RPS14. Consistent with disruption of the assembly and function of the small ribosomal subunit, KO and KI/-HSPC exhibited rapid and robust impairment of global protein translation. Polysome profiling indicated an increase in monosomes and a decrease in polysomes in KO cells, consistent with an inability of ribosomes to initiate translation and move along the mRNA. To determine the role of the translation defect in the cell growth deficiency of DDX41-deficient cells, we treated WT, HET, and KO HSPC with the translation inhibitor puromycin and determined that KO cells were relatively more sensitive to translation inhibition, indicating that DDX41-deficient cells are specifically sensitive to further reduction in protein translation. This data supports the conclusion that the cell lethality caused by DDX41 loss is related to ribosome dysfunction. Mechanistically, we find that DDX41-deficient cells have stalled ribosomal RNA (rRNA) processing, characterized by increased unprocessed rRNA and decreased processed rRNA intermediates. In conclusion, we identify a novel function of DDX41 in regulating rRNA processing and ribosome formation that is essential for the survival and proliferation of HSPC. The loss of DDX41 may contribute to MDS as a result of impaired ribosome function, as has been previously reported in patients bearing mutations in other ribosome regulators. Disclosures Starczynowski: Kurome Therapeutics: Consultancy.

The development of complex organisms requires the formation of diverse cell types from common stem and progenitor cells. GATA family transcriptional regulators and their dedicated co-factors, termed Friend of GATA (FOG) proteins, control cell fate and differentiation in multiple tissue types from Drosophila to man. FOGs can both facilitate and antagonize GATA factor transcriptional regulation depending on the factor, cell, and even the specific gene target. In this review, we highlight recent studies that have elucidated mechanisms by which FOGs regulate GATA factor function and discuss how these factors use these diverse modes of gene regulation to control cell lineage specification throughout metazoans.

GATA-1 and its cofactor FOG-1 are required for the differentiation of erythrocytes and megakaryocytes. In contrast, mast cell development requires GATA-1 and the absence of FOG-1. Through genome-wide comparison of the chromatin occupancy of GATA-1 and a naturally occurring mutant that cannot bind FOG-1 (GATA-1(V205G)), we reveal that FOG-1 intricately regulates the chromatin occupancy of GATA-1. We identified GATA1-selective and GATA-1(V205G)-selective binding sites and show that GATA-1, in the absence of FOG-1, occupies GATA-1(V205G)-selective sites, but not GATA1-selective sites. By integrating ChIP-seq and gene expression data, we discovered that GATA-1(V205G) binds and activates mast cell-specific genes via GATA-1(V205G)-selective sites. We further show that exogenous expression of FOG-1 in mast cells leads to displacement of GATA-1 from mast cell-specific genes and causes their downregulation. Together these findings establish a mechanism of gene regulation whereby a non-DNA binding cofactor directly modulates the occupancy of a transcription factor to control lineage specification.

There are many examples of transcription factor families whose members control gene expression profiles of diverse cell types. However, the mechanism by which closely related factors occupy distinct regulatory elements and impart lineage specificity is largely undefined. Here we demonstrate on a genome wide scale that the hematopoietic GATA factors GATA-1 and GATA-2 bind overlapping sets of genes, often at distinct sites, as a means to differentially regulate target gene expression and to regulate the balance between proliferation and differentiation. We also reveal that the GATA switch, which entails a chromatin occupancy exchange between GATA2 and GATA1 in the course of differentiation, operates on more than one-third of GATA1 bound genes. The switch is equally likely to lead to transcriptional activation or repression; and in general, GATA1 and GATA2 act oppositely on switch target genes. In addition, we show that genomic regions co-occupied by GATA2 and the ETS factor ETS1 are strongly enriched for regions marked by H3K4me3 and occupied by Pol II. Finally, by comparing GATA1 occupancy in erythroid cells and megakaryocytes, we find that the presence of ETS factor motifs is a major discriminator of megakaryocyte versus red cell specification.

The major circulating form of vitamin D, 25-hydroxycholecalciferol (25D3), circulates bound to vitamin D-binding protein (DBP). Prior to activation to 1,25-dihydroxycholecalciferol in the kidney, the 25D3-DBP complex is internalized via receptor-mediated endocytosis, which is absolutely dependent on the membrane receptors megalin and cubilin and the adaptor protein disabled-2 (Dab2). We recently reported that mammary epithelial cells (T-47D) expressing megalin, cubilin, and Dab2 rapidly internalize DBP via endocytosis, whereas cells that do not express all 3 proteins (MCF-7) do not. The objectives of this study were to characterize megalin, cubilin, and Dab2 expression and transport of DBP in human mammary epithelial cells. Using immunoblotting and real-time PCR, we found that megalin, cubilin, and Dab2 were expressed and dose dependently induced by all-trans-retinoic acid (RA) in T-47D human breast cancer cells and that RA-treated T-47D cells exhibited enhanced DBP internalization. These are the first studies to our knowledge to demonstrate that mammary epithelial cells express megalin, cubilin, and Dab2, which are enhanced during differentiation and may explain, at least in part, our finding that receptor-mediated endocytosis of DBP is upregulated in differentiated mammary epithelial cells.

DNA repair plays a crucial role in embryonic and somatic stem cell biology and cell reprogramming. The Fanconi anemia (FA) pathway, which promotes error-free repair of DNA double-strand breaks, is required for somatic cell reprogramming to induced pluripotent stem cells (iPSC). Thus, cells from Fanconi anemia patients, which lack this critical pathway, fail to be reprogrammed to iPSC under standard conditions unless the defective FA gene is complemented. In this study, we utilized the oncogenes of high-risk human papillomavirus 16 (HPV16) to overcome the resistance of FA patient cells to reprogramming. We found that E6, but not E7, recovers FA iPSC colony formation and, furthermore, that p53 inhibition is necessary and sufficient for this activity. The iPSC colonies resulting from each of these approaches stained positive for alkaline phosphatase, NANOG, and Tra-1-60, indicating that they were fully reprogrammed into pluripotent cells. However, FA iPSC were incapable of outgrowth into stable iPSC lines regardless of p53 suppression, whereas their FA-complemented counterparts grew efficiently. Thus, we conclude that the FA pathway is required for the growth of iPSC beyond reprogramming and that p53-independent mechanisms are involved. IMPORTANCE A novel approach is described whereby HPV oncogenes are used as tools to uncover DNA repair-related molecular mechanisms affecting somatic cell reprogramming. The findings indicate that p53-dependent mechanisms block FA cells from reprogramming but also uncover a previously unrecognized defect in FA iPSC proliferation independent of p53.

Children with Down syndrome develop a unique congenital clonal megakaryocytic proliferation disorder (transient myeloproliferative disorder [TMD]). It is caused by an expansion of fetal megakaryocyte-erythroid progenitors (MEPs) triggered by trisomy of chromosome 21 and is further enhanced by the somatic acquisition of a mutation in GATA1. These mutations result in the expression of a short-isoform GATA1s lacking the N-terminal domain. To examine the hypothesis that the Hsa21 ETS transcription factor ERG cooperates with GATA1s in this process, we generated double-transgenic mice expressing hERG and Gata1s. We show that increased expression of ERG by itself is sufficient to induce expansion of MEPs in fetal livers. Gata1s expression synergizes with ERG in enhancing the expansion of fetal MEPs and megakaryocytic precursors, resulting in hepatic fibrosis, transient postnatal thrombocytosis, anemia, a gene expression profile that is similar to that of human TMD and progression to progenitor myeloid leukemia by 3 months of age. This ERG/Gata1s transgenic mouse model also uncovers an essential role for the N terminus of Gata1 in erythropoiesis and the antagonistic role of ERG in fetal erythroid differentiation and survival. The human relevance of this finding is underscored by the recent discovery of similar mutations in GATA1 in patients with Diamond-Blackfan anemia.

The transcription factor Ikaros regulates the development of hematopoietic cells. Ikaros-deficient animals fail to develop B cells and display a T-cell malignancy, which is correlated with altered Notch signaling. Recently, loss of Ikaros was associated with progression of myeloproliferative neoplasms to acute myeloid leukemia and increasing evidence shows that Ikaros is also critical for the regulation of myeloid development. Previous studies showed that Ikaros-deficient mice have increased megakaryopoiesis, but the molecular mechanism of this phenomenon remains unknown. Here, we show that Ikaros overexpression decreases NOTCH-induced megakaryocytic specification, and represses expression of several megakaryocytic genes including GATA-1 to block differentiation and terminal maturation. We also demonstrate that Ikaros expression is differentially regulated by GATA-2 and GATA-1 during megakaryocytic differentiation and reveal that the combined loss of Ikzf1 and Gata1 leads to synthetic lethality in vivo associated with prominent defects in erythroid cells and an expansion of megakaryocyte progenitors. Taken together, our observations demonstrate an important functional interplay between Ikaros, GATA factors, and the NOTCH signaling pathway in specification and homeostasis of the megakaryocyte lineage.

The basic-helix-loop-helix transcription factor HeyL is expressed at high levels by neural crest progenitor cells (NCPs) that give rise to neurons and glia in dorsal root ganglia (DRG). Since HeyL expression was observed in these NCPs during the period of neurogenesis, we generated HeyL null mutants to help examine the factor's role in ganglion neuronal specification. Homozygous null mutation of HeyL reduced the number of TrkC+ neurons in DRG at birth including the subpopulation that expresses the ETS transcription factor ER81. Conversely, null mutation of the Hey paralog, Hey1, increased the number of TrkC+ neurons. Null mutation of HeyL increased expression of the Hey paralogs Hey1 and Hey2, suggesting that HeyL normally inhibits their expression. Double null mutation of both Hey1 and HeyL rescued TrkC+ neuron numbers to control levels. Thus, the balance between HeyL and Hey1 expression regulates the differentiation of a subpopulation of TrkC+ neurons in the DRG.

Objective It has been demonstrated that high concentration of the transcription factor PU.1 (encoded by Sfpi1 ) promotes macrophage development, whereas low concentration induces B-cell development in vitro. This has led to the hypothesis that lower levels of PU.1 activity are required for B cell than for macrophage development in vivo. We utilized an allele of Sfpi1 (termed BN) with a mutation in the first coding exon, which resulted in a reduction of PU.1 expression in order to test this hypothesis. Materials and Methods Using gene targeting in embryonic stem cells, two ATG-start site codons of PU.1 were mutated, resulting in reduced PU.1 expression originating from a third start codon. Mice were assayed for phenotypic abnormalities using fluorescence-activated cell sorting, microscopy, and colony-forming ability. In addition, isolated cells were tested for their differentiation potential in vitro and in vivo. Results Lymphoid and myeloid cells derived from cultured Sfpi1 BN/BN fetal liver cells had reduced levels of PU.1 expression and activity. B-cell development was intrinsically blocked in cells isolated from Sfpi1 BN/BN mice. In addition, myeloid development was impaired in Sfpi1 BN/BN fetal liver. However, neonatal Sfpi1 BN/BN mice had a dramatic expansion and infiltration of immature myeloid cells. Conclusion Contrary to our original hypothesis, high levels of PU.1 activity are required to induce both myeloid and B-cell development. In addition, neonatal mice homozygous for the hypomorphic allele acquire a myeloproliferative disorder and die within 1 month of age.

The Fanconi Anemia (FA) DNA Repair pathway functions through homologous recombination for error-free repair of DNA interstrand crosslinks. Loss of function of this pathway causes a complex genetic disease that is characterized by congenital abnormalities, bone marrow failure (BMF), and extreme incidence of squamous cell carcinomas. BMF is caused by exhaustion of hematopoietic stem and progenitor cells (HSPCs) and is nearly 100% penetrant by age 40 in FA patients, indicating a profound sensitivity of HSPCs to FA pathway deficiency. In contrast, stem cells in other rapidly regenerating tissues, such as the skin and intestine, are not similarly exhausted. Interestingly, squamous epithelium is highly prone to transformation while intestinal epithelium is not. In order to explore the developmental origins of such striking tissue-specific phenotypes in FA patients, we have generated induced pluripotent stem cell (iPSC) lines conditional for FA pathway function (cFA-iPSCs) and used them to derive FA-proficient and deficient in vitro models of diverse tissues. FA patient cells are refractory to reprogramming. To circumvent this defect and prevent the selection of FA-resistant iPSC clones, fibroblasts from 2 FANCA patients were inducibly complemented with a FANCA transgene under the control of a tetracycline-inducible promoter and then were reprogrammed to iPSC. In this way, the FA pathway was functional throughout reprogramming and could then be turned on or off in established iPSC lines by the addition or withdrawal of doxycycline (DOX) to the media. Here, we describe the effect of FA pathway loss on iPSCs, and present preliminary data on iPSC-derived equivalents of three lineages: hematopoietic, squamous, and intestinal. First, functional consequences of FA pathway loss on iPSC pluripotency and self-renewal were examined. Upon withdrawal of DOX from the culture media, the complementing FA transgene was effectively silenced, resulting in loss of FA pathway function within 7 days. FA-deficient iPSCs maintained normal expression of OCT-3/4 and NANOG and formed teratomas in NSG mice, indicating that pluripotency was maintained. However, profound cell cycle arrest and apoptosis were observed under normal in vitro culture conditions within 7 days of DOX-withdrawal, and the iPSCs failed to expand by 2-3 passages. Thus, we concluded that iPSCs require an intact FA pathway for self-renewal in vitro. Mechanistic studies of FA pathway-deficient iPSCs revealed a 10-fold increase in gH2AX foci in the G2-M phase of the cell cycle. This correlated with activated DNA damage response signaling through ATR and CHK1. Inhibition of CHK1 completely restored the growth of FA-deficient iPSCs to that of their FA-proficient counterparts through a remarkable rapid bypass of the G2-M checkpoint. Unexpectedly, cells maintained in CHK1 inhibitor for over 40days accrued few karypotypic abnormalities ( We next differentiated the cFA-iPSCs into 3D squamous epithelium and intestine with timed-withdrawal of the FA pathway to determine the effect of FA pathway loss on tissue development and homeostasis. Squamous epithelial rafts and intestinal organoids were generated in the presence and absence of DOX using established protocols. These demonstrated that FA pathway loss does not cause gross abnormalities in epithelial tissues, in line with patient phenotypes. We will present the latest results on proliferation, survival, and differentiation of stem and progenitor cells within each tissue. These will include an assessment of epithelial hyperplasia, which has been observed previously in immortalized FA patient keratinocyte-derived organotypic squamous epithelial rafts and in the oral epithelia of FANCD2 knockout mice. Finally, we will present our latest data on the effects of FA deficiency on hematopoietic progenitor cells, which are currently being generated from the cFA-iPSCs. Collectively, these experiments will quantify cell intrinsic sensitivity of pluripotent versus somatic stem cells that reside in diverse tissue types to loss of the FA pathway. The results may inform the development of novel therapeutics to treat FA BMF without increasing disease risk in other tissues. Disclosures No relevant conflicts of interest to declare.

As the source of all cells in the developing embryo proper, embryonic stem cells (ESC) bear the unique responsibility to prevent mutations from being propagated throughout the entire organism and the germ line. It is likely for this reason that ESC and induced pluripotent stem cells (iPSC) maintain a dramatically lower mutation frequency than cultured somatic cells. Multiple mechanisms for this enhanced genomic surveillance have been proposed, including hypersensitivity of DNA damage response signaling pathways and increased activity of error-free DNA repair pathways, such as homologous recombination. However, the effect of loss of function of DNA repair pathways in these cells remains poorly understood. The Fanconi Anemia (FA) pathway is a DNA repair pathway that is required for the repair of DNA interstrand crosslink damage and also promotes repair of DNA double-strand breaks by homologous recombination . Genetic defects in this pathway cause a disease characterized by bone marrow failure and extreme cancer incidence. Several recent studies have revealed that the FA pathway is required for efficient somatic cell reprogramming to iPSC and suggest that FA cells undergo cell death during this process. Another recent study found that the growth of FA patient-specific iPSC was attenuated with a G2/M arrest when compared to control iPSC, suggesting that these cells arrest upon failed DNA repair. In this study, we sought to determine the effects of acute loss of function of the FA pathway in iPSC through the generation of FA patient-derived iPSC with inducible complementation of the defective FA gene. Fibroblasts were cultured from skin biopsies of multiple FA patients and transduced with a lentiviral vector expressing the complementing FA gene product under DOX-inducible control. Cells were then reprogrammed to iPSC using episomal transfection. These cells formed iPSC colonies only when reprogramming was carried out in the presence of DOX, confirming that the FA pathway is required for efficient reprogramming. Once cell lines were obtained, DOX-dependent FA functionality was verified based on FANCD2 monoubiquitination and nuclear focus formation after treatment with DNA damaging agents. We then cultured the iPSC for extended periods of time in the presence and absence of DOX. Interestingly, the cultures underwent profound cell death and cell cycle arrest within 7 days of DOX-withdrawal and completely failed to expand after one passage. EdU cell cycle analysis confirmed cell cycle arrest in the G2/M phase. Furthermore, cleaved caspase 3 staining confirmed that the number of apoptotic cells increased by 3-fold in the -DOX culture. Despite these effects, cells cultured in both the presence and absence of DOX formed teratomas in nude mice, thus indicating the maintenance of full differentiation capacity in the absence of the FA pathway. In order to determine the mechanisms underlying G2/M arrest and cell death, expression of p53 and its target genes was detected by both western blot analysis and qRT-PCR. Only a slight increase in p53 activation was observed by 7 days post DOX-withdrawal. Furthermore, knockdown of p53 resulted in rescue from apoptosis to normal levels but not rescue from cell cycle arrest. Increased ATM and ATR DNA damage sensor kinase activities were also detected in –DOX cells, concominant with increased phosphorylation of the ATM-target Chk2 and reduced abundance of the G2/M checkpoint protein CDC25A. These results reveal hyperactive DNA damage responses upon FA loss which may underlie the attenuated cell cycle progression of FA-iPSC independent of p53. Remarkably, effects in this FA model system appear equivalent to those responsible for the depletion of HSC in the bone marrow of FA patients. Thus, iPSC models may be useful for future studies of the mechanisms underlying FA stem cell arrest and for the development of therapeutics that alleviate these phenotypes. Disclosures No relevant conflicts of interest to declare.

The X-linked blood transcription factor GATA1 is required for the survival and maturation of erythroid cells. Loss of Gata1 causes profound anemia and related mid-gestation lethality in mouse models and human GATA1 mutations are associated with congenital dyserythropoietic anemia, congenital thrombocytopenia, porphyria, Diamond-Blackfan anemia (DBA) and acute megakaryoblastic leukemia in children with Down syndrome (DS-AMKL). In DBA, DS-AMKL, and a subset of congenital anemia, GATA1 mutations cause the exclusive production of a shorter GATA1 isoform, termed GATA1s. The GATA1s isoform retains both zinc fingers and binds DNA and cofactors accordingly, but lacks the N-terminal 83 residues of full-length GATA1. Intriguingly, exclusive production of GATA1s promotes AMKL in the context of Down syndrome (trisomy 21), while disomic individuals harboring the same type of mutations suffer from anemia and severe red cell defects. A mutation causing exclusive production of Gata1s in mice does not appear to affect adult hematopoiesis, but has been shown to cause a marked expansion of megakaryocytes in the fetal liver. The effects of the replacement of Gata1 with Gata1s during fetal liver erythropoiesis, however, remain uncharacterized. To investigate the effects of exclusive Gata1s production during fetal hematopoiesis, we performed comprehensive phenotypic and mechanistic studies using Gata1s knock-in embryos. We found significant changes in myelo-erythroid differentiation beyond the known expansion of megakaryocytes. Flow cytometric analysis revealed altered erythroid differentiation at embryonic days 12.5 and 14.5, evidenced by decreased Ter119 and increased CD71 expression, characteristic of delayed erythroid maturation. Changes in expression of myelo-erythroid progenitor commitment markers were also discovered. Specifically, we observed marked decreases in pre-CFU-E and CFU-E committed progenitors and an increase in pre-GM and pre-MegE populations. Our findings are consistent with a bias towards megakaryopoiesis at the expense of erythroid commitment caused by the expression of Gata1s in place of full-length Gata1. A shift in differentiation was also observed in the embryonic granulocyte/ macrophage lineage, with an increased generation of macrophages with fewer developing granulocytes. Mechanistically, we found that expression of erythroid -specific Gata1 target genes such as Alas2, Slc4a1 and Klf1 are markedly reduced in the Gata1 knock-in erythroid cells, indicating that Gata1s is a less effective transcriptional activator than full-length Gata1. In particular, given that Klf1 functions to promote erythroid specification downstream of the megakaryocyte-erythrocyte progenitor, our results suggest that Gata1s may promote megakaryocyte differentiation at the expense of erythroid differentiation, in part, by failing to activate Klf1. Taken together, our studies demonstrate a stage-specific requirement for full-length Gata1 during embryonic erythropoiesis. Furthermore, erythroid defects associated with exclusive production of Gata1s in humans may result from an incomplete activation of the erythroid transcriptional program. Disclosures: Crispino: Sanofi: Research Funding.

Abstract 3451 Down syndrome (DS) is associated with an increased frequency of Acute Megakaryoblastic Leukemias (AMKL). In the context of DS, AMKL is usually preceded by the development of a transient myeloproliferative disorder (TMD). Up to 10% of DS children may develop this disorder, and approximately 20–30% of those with TMD go on to develop AMKL, which may or may not be preceded by a return to normal hematopoiesis. While trisomy 21 (tri21) alone is not sufficient to develop AMKL, it is clear that the acquisition of somatic mutations in GATA1 is a requirement for the development of DS-AMKL. Much has been studied in relation with this genetic mutation, however not much is known about the epigenetic changes accompanying the development of AMKL in DS patients. Given the step-wise nature of the development of DS-AMKL, we hypothesized that distinct and cumulative epigenetic changes would be associated with each of the phases of the disease. In order to address this hypothesis we performed DNA methylation profiling of a cohort of 7 primary DS-AMKL samples using the latest design of the HELP microarray assay, which covers ∼230,000 CpG sites at ∼22,000 RefSeq promoter regions. In order to better understand how aberrant epigenetic patterns become established during the development of this disease we compared these DNA methylation profiles to those of mononuclear cells from normal fetal liver (FL-MNC) (n=4), tri21 FL-MNC (n=4) and TMD blasts (n=6). We identified a set of 711 unique genes that displayed significant loss of methylation in Tri21 FL-MNC with respect to control FL-MNC (FDR < 5% and methylation delta ≥ 25%). However, when tri21 FL-MNC were compared to the GATA1s positive TMD blasts, we observed that TMD samples acquired significant hypermethylation of 346 unique genes at the same significance cutoff. A direct comparison of the genes affected by these two opposing waves of methylation changes demonstrated that different sets of genes were being targeted at each stage. Next we compared the DNA methylation profiles of TMD and DS-AMKL blasts, and found them to be virtually identical, indicating that an epigenetic imprint becomes established at the TMD phase, which is still present at the leukemic phase. Finally we compared DS-AMKL to a cohort of 8 non-DS AMKL, and observed that DS-AMKL samples display significant hypomethylation of 267 unique genes, indicating that these two diseases are not only genetically but also epigenetically distinct. In determining the location of the epigenetic changes acquired at the different stages of the disease we found that, similar to what had been previously reported for gene expression, DNA methylation changes did not localize solely to chr 21, but that these changes were distributed along all chromosomes. However, detailed analysis of chr 21 revealed that changes on this chromosome preferentially targeted a specific region on the long arm, the Down Syndrome Critical Region (DSCR). This region becomes significantly hypomethylated in Tri21 FL-MNC and remains hypomethylated through all the stages of the disease. Several groups have sought to identify candidate genes on the DSCR that may contribute to the pathogenesis of the disease. Two genes, BACH1 and CXADR, have been identified in common by two different studies as being overexpressed in DS-AMKL vs. Non-DS AMKL. Both of these genes displayed aberrant hypomethylation at their promoter regions in Tri21 FL MNC, TMD and DS-AMKL samples: such hypomethylation was absent from all the control samples. Finally, given that GATA1 mutations are a requirement for the development of DS-AMKL, we compared the different aberrant DNA methylation signatures to the normal GATA1 targets identified by ChIP sequencing experiments, and found 20–25% overlap. In summary, we have completed the first comprehensive DNA methylation study of the developmental phases of DS-AMKL and have demonstrated that a) specific epigenetic changes are associated with the different stages of the disease, b) these changes occur in ‘waves' of sequential hypo and hypermethylation, c) aberrant DNA methylation on chr 21 preferentially targets the DSCR and results in aberrant overexpression of the associated genes, and d) epigenetic profiles of TMD and DS-AMKL are virtually identical. These observations lead us to believe that an epigenetic memory established at the TMD stage may be at least in part contributing to the development of full-blown AMKL at a later time point. Disclosures: No relevant conflicts of interest to declare.

Abstract 2604 GABP transcription factor has been implicated in the regulation of genes that are required for normal megakaryocytic differentiation. Megakaryocytes express several related ets factors, including Fli-1, ets2, and GABP, and it has been unclear if any single ets factor plays a non-redundant role in these cells. The tetrameric GABP transcription factor complex contains two molecules of GABPα, which binds DNA, and two molecules of GABPβ, which encodes the transcription activation domain. We created mice with loxP recombination sites which flank exons that encode Gabpa ets-related DNA-binding domains (floxed Gabpa, or Gabpa fl/fl), and bred them to mice that carry Mx1-Cre. In response to injection with the synthetic polynucleotide, pIC, these mice express Cre recombinase and efficiently delete Gabpa; these animals are referred to as knock-out (KO) mice. Control mice, which carry floxed Gabpa but lack Mx1-Cre, were treated identically with pIC. Platelet counts of KO mice declined to less than 50,000 within nine days, while platelet counts in control mice were unaffected. One half of KO mice died within two weeks of Gabpa deletion due to widespread visceral hemorrhage. Histologic examination of the bone marrow and spleen reveals a loss of megakaryocytes in KO mice, compared to control animals. Residual megakaryocytes in KO mice exhibit increased expression of platelet-specific antigens, CD41 and CD42, and a significant increase of DNA ploidy. Because Gabpa KO mice died with a striking loss of megakaryocytes and platelets, yet megakaryocytic differentiation appeared to be unimpaired, we sought to better define the nature of this defect. Bone marrow from Gabpa fl/fl mice was infected with a retrovirus that expresses Cre and green fluorescent protein (GFP), or control virus that expresses only GFP; grown for three days in liquid culture conditions that foster megakaryocytic differentiation; and analyzed for CD41 and CD42 expression, ploidy, and apoptosis. Gabpa was efficiently deleted by the Cre-bearing virus, and Gabpa deletion was associated with increased expression of CD41 and CD42, and increased DNA ploidy. However, Gabpa deletion was also associated with increased megakaryocytic-associated apoptosis, and in vitro megakaryocyte colony formation was dramatically reduced in Gabpα null cells. In summary, deletion of Gabpa in mice is associated with plummeting platelet counts, widespread visceral hemorrhage, and a loss of splenic and bone marrow megakaryocytes. In vitro analysis demonstrates intact megakaryocytic differentiation and a profound loss of megakaryocytic progenitor cells. The increased expression of megakaryocytic antigens and DNA ploidy may indicate that Gabpa deletion enhances megakaryocytic differentiation or, alternatively, it may represent selective loss of more immature megakaryocytic cells following Gabpa disruption. Data that directly test these alternative hypotheses will be presented. In summary, we demonstrate that GABP plays a non-redundant role in megakaryocyte development, that GABP is required for the proliferation of committed megakaryocytic progenitors, but that GABP is not required for the later stages of megakaryocytic maturation. Disclosures: No relevant conflicts of interest to declare.

Abstract 3643 GATA-1 is a zinc finger transcription factor that regulates the differentiation of megakaryocytes and erythrocytes from the megakaryocyte-erythrocyte progenitor (MEP). Mutations in GATA1 are associated with hematologic malignancies of these two related lineages. Acquired mutations that lead to expression of the short isoform of GATA-1, termed GATA-1s, are associated with Acute Megakaryocytic Leukemia in children with Down syndrome (DS-AMKL). Moreover, inherited mutations in the N-finger of GATA1, such as V205M, cause a set of related diseases characterized by dyserythropoietic anemia and thrombocytopenia. These latter mutations disrupt recruitment of the essential cofactor FOG-1 and thus promote disease by interfering with the ability of the GATA-1:FOG complex to properly regulate gene expression. Despite the fact that V205 lies along the surface of the zinc finger opposite to DNA, previous studies suggest that FOG-1 may regulate the chromatin binding activity of GATA-1 at a subset of sites. In contrast to V205 mutation, the precise mechanisms by which GATA-1s contributes to disease is poorly understood. Previous studies have shown that GATA-1s uncouples megakaryocyte proliferation from differentiation, likely by an inability of GATA-1s to properly repress expression of a subset of GATA-1 target genes. We hypothesized that both inherited and acquired GATA1 mutations contribute to disease by interfering with not only target gene activation or repression, but also with GATA-1 chromatin binding. In order to define the chromatin binding activity of GATA-1 and its disease associated mutants, we performed chromatin immunoprecipitation coupled with next generation sequencing (ChIP-Seq) for wild-type GATA-1, GATA-1s and GATA-1V205G in the G1ME cell line. G1ME cells, which were derived from GATA-1 null ES cells, approximate an MEP in that they can differentiate into either erythroid cells (in the presence of EPO) or megakaryocytes (in the presence of TPO) upon reconstitution with GATA-1. We expressed GATA-1, GATA-1s, or GATA-1V205G in G1ME cells by retroviral transduction and subjected the cells to ChIP for GATA-1. The resulting DNA was sequenced on the Illumina GAII, yielding 11.8M, 10.4M, and 8.5M uniquely mapped reads. Analysis of the datasets using QuEST yielded 2367, 963, and 4130 peaks, respectively, with an FDR of Disclosures: No relevant conflicts of interest to declare.

Abstract 469 Children with Down syndrome (DS) have a 500-fold increased risk of Acute Megakaryoblastic Leukemia (DS-AMKL). In addition to trisomy 21, DS-AMKL blasts harbor somatic mutations in the gene encoding GATA-1, an essential transcriptional regulator of erythroid and megakaryocytic differentiation. In every case, the GATA1 mutations lead to a block in expression of the full-length protein, but allow for the translation of a shortened isoform named GATA-1s, which lacks the N-terminal transcriptional activation domain. Recent studies have shown that mutations in of JAK2, JAK3 and MPL are another feature of AMKL. Whereas the impact on hematopoiesis of individual GATA-1s, JAK2 V617F and MPL W515L mutants is relatively well known, the specific effects of trisomy 21, the initiating event in DS leukemogenesis, remains unclear. Here, we used two different experimental models to identify specific genes on human chromosome 21 (Hsa21) that participate in leukemogenesis: human DS-AMKL cell lines and the Ts1Rhr mouse strain, which is trisomic for only 33 orthologs of Hsa21 genes. Comprehensive analysis of hematopoiesis in progressively more complex mouse models of DS disorders, including Ts1Rhr mice, Ts1Rhr/KI-Gata-1s compound mutant mice, and recipients of Ts1Rhr/KI-Gata-1s hematopoietic progenitors expressing MPL W515L, revealed that the presence of trisomy 21 impacts the phenotype of each genetic background. Ts1Rhr mice develop a progressive myeloproliferative disease with megakaryocytic features while Ts1Rhr/KI-Gata1s double transgenic mice develop thrombocytosis at an earlier age compared to Gata1s mutant or Ts1Rhr mice alone. However neither the Ts1Rhr nor the compound mutant mice develop AMKL. In stark contrast, expression of MPL W515L in Ts1Rhr/KI-Gata1s hematopoietic progenitors led to a rapid lethal megakaryoblastic disorder characterized by profound marrow and splenic fibrosis and the presence of immature megakaryoblasts in lethally irradiated recipients. Of note, transplantation of MPL W515L expressing singly mutant Ts1Rhr or KI-Gata-1s progenitors failed to cause a similar disease. These findings show that three oncogenic events are necessary and sufficient to induce a megakaryoblastic leukemia in recipient mice. In parallel, to identify specific genes on Hsa21 that contribute to AMKL, we performed shRNA screening in DS-AMKL cell lines by knocking down the human orthologs of the 33 genes trisomic in the Ts1Rhr mice. We then evaluated the consequence of knockdown on megakaryocyte proliferation, survival and/or differentiation. This study revealed that altered expression of ERG, DYRK1A, CHAF1B and HLCS affected megakaryocyte growth. Analysis of their expression in human samples suggest that these four genes likely play a role in etiology of DS-AMKL. Moreover expression evaluation of those genes in our in vivo models of 1, 2 or 3 genetic events revealed that ERG, a known megakaryoblastic oncogene, and DYRK1A are the most prominent genes implicated in these megakaryocytic disorders. DYRK1A (dual specificity tyrosine-regulated kinase 1A) has been functionally implicated in many developmental disorders associated with DS and shown to be constitutively phosphorylated/activated in human AMKL cell lines. We found that overexpression of DYRK1A cooperates with Gata1s to lead to a increase in megakaryocyte growth in liquid culture and in colony assays in vitro. In summary, we show that partially trisomic Ts1Rhr mice display progressive defects in the megakaryocytic compartment, that mutagenesis of Gata1 in the Ts1Rhr background exacerbates the Ts1Rhr phenotype, and that addition of an activating mutation of MPL leads to a fulminant megakaryocytic leukemia with myelofibrosis. Moreover, among the 33 genes present in 3 copies in the Ts1Rhr background, we identify DYRK1A as a likely megakaryoblastic oncogene in DS-AMKL. Disclosures: No relevant conflicts of interest to declare.

Abstract 2605 GATA family transcription factors play critical roles in various mammalian developmental processes, including hematopoiesis. In particular, GATA-1 expression is necessary for proper terminal differentiation of mast cells, red blood cells, eosinophils, and megakaryocytes. GATA-2 is required for proliferation and survival of hematopoietic stem and progenitor cells, and is also expressed in erythroid precursors, mast cells, and early megakaryocytes. In developing erythrocytes, GATA-2 and GATA-1 are responsible for temporal control of a multi-factor transcriptional regulatory network that involves (a) GATA-2 positively regulating its own gene transcription, (b) GATA-2 positively regulating the expression of the Gata1 gene, (c) GATA-1 positively regulating its own gene transcription, and (d) GATA-1 negatively regulating Gata2 gene transcription. During this sequence of events, a “GATA switch” occurs, wherein GATA-1 replaces GATA-2 at canonical GATA binding sites within the regulatory regions of the Gata2 and Gata1 genes, as well as at many other genomic loci that encode genes responsible for proliferation or differentiation of erythroid progenitors. Similarly, in early megakaryocytic progenitors, GATA-2 promotes proliferation and suppresses expression of alternative-lineage genes; subsequent activation of GATA-1 precipitates terminal differentiation with concomitant downregulation of proliferative genes and activation of megakaryocyte-specific genes. The presence or role of a GATA switch in megakaryocytes has not yet been formally investigated. To address the role of the GATA switch in megakaryocytic differentiation, we performed massively parallel sequencing of chromatin immunoprecipitation (ChIP-Seq) material for GATA-2 and GATA-1 before or after GATA-1 restoration in the GATA1-null megakaryocytic progenitor cell line, G1ME. We obtained 22 million unique GATA-2 tags and 10 million unique GATA-1 tags and identified 14985 and 5102 high-confidence GATA-2 and GATA-1 binding sites, respectively. Additionally, we used 13 million tags from ChIP for H3K4me3 to identify 24909 genomic sites enriched for the presence of trimethylated lysine-4 on histone H3. Trimethylated H3K4 marks nearly half of all GATA-1 bound sites and one-third of GATA-2 bound sites. Over 40% of the sites bound by GATA-1 in differentiating G1ME cells were also bound by GATA-2 in proliferating G1ME cells, indicating that a GATA switch does indeed occur during megakaryocyte development. Coordinated analyses of these occupancy data with previously published gene expression datasets show that the lists of bound genes are significantly enriched for differentially expressed genes and the data depict a generally antagonistic relationship between GATA-2 and GATA-1. Interestingly, we find that even among genes that don't contain GATA switch sites, greater than 40% of those bound by GATA-1 were also occupied by GATA-2 at distinct sites. To further characterize the occupied loci, we surveyed the genomic regions bound by GATA-1 and GATA-2 to detect motifs enriched in the sequences surrounding the peak calls. As expected, we found that over 80% contained the canonical WGATAR binding motif. In contrast to reports of motifs enriched in GATA-1 ChIP studies in erythroid cells, we failed to observe significant enrichment of LRF binding motifs. Rather, the GATA-1 and GATA-2 bound regions in megakaryocytes are strongly enriched for motifs that match the binding sites for Ets family transcription factors. Finally, we have found that these genomic regions are indeed occupied by one or more Ets factors in proliferating G1ME cells. Together, these data establish the presence of a GATA switch in megakaryocyte development and provide novel insights into coordinated gene regulation by GATA factors and the differences between the closely related erythroid and megakaryocyte lineages. Disclosures: No relevant conflicts of interest to declare.

Abstract 1602 The hematopoietic transcription factor Ikaros regulates the development of lymphoid cells. In particular, Ikaros has been shown to suppress expression of Notch target genes in developing thymocytes. In addition, Ikaros-deficient animals fail to develop B cells and instead display a T cell lymphoproliferative disorder. Furthermore, alterations in Ikaros gene expression are associated with human lymphoid neoplasms and loss of Ikaros is associated with progression of myeloproliferative neoplasms to acute myeloid leukemia. However, the role of Ikaros in normal and malignant myelopoiesis is not well characterized. Therefore, we first analyzed myeloid development in Ikaros deficient mice and observed extramedullary hematopoiesis in the spleen and a striking thrombocytosis in the peripheral blood of 8–9 week-old Ikaros-null mice (2×106 platelets/μl, compared to 0.5×106 platelets/μl in wild-type littermates). Flow cytometry, histology and colony forming assays revealed that the bone marrow and spleen of young Ikaros-null mice harbored a substantial increase in the numbers of megakaryocytes and myeloid cells. We next investigated how Ikaros activity is reduced during terminal differentiation. Previous reports have shown that GATA1-deficient and GATA1s mutant megakaryocytes, which are associated with Down syndrome acute megakaryoblastic leukemia (DS-AMKL), express aberrantly high levels of Ikaros, suggesting that Ikaros is a target of GATA-1 repression during terminal megakaryocyte differentiation. By chromatin immunoprecipitation assays, we found multiple sites in the Ikaros locus that are bound by GATA-2 in proliferating progenitors and by GATA-1 in maturing megakaryocytes. Furthermore, we discovered that GATA-1s fails to occupy these conserved Ikaros loci. Together these results strongly suggest that GATA-1, but not GATA-1s, displaces GATA-2 from the Ikaros gene during differentiation and that this GATA switch leads to repression of Ikaros, in turn allowing for terminal maturation of megakaryocytes. Since Notch participates in specification of the megakaryocyte lineage, we then asked whether Ikaros inhibited megakaryopoiesis by interfering with Notch signaling. Retroviral transduction of full-length Ikaros in murine common myeloid progenitors, but not in LSK cells, reduced megakaryocyte development upon Notch stimulation in OP9-DL1 stromal co-cultures. Ectopic expression of Ikaros in the 6133 cell line, which models the t(1;22) subtype of AMKL and shows aberrant Notch target genes activation by the OTT-MAL fusion oncogene, inhibited proliferation and induced apoptosis. Genome wide expression profiling in transduced 6133 cells confirmed that Ikaros alters expression of several genes involved in the Notch and growth factor signaling pathways. These results indicate that Ikaros restricts megakaryocyte development and inhibits proliferation of OTT-MAL-transformed AMKL cells at least in part by suppressing Notch signaling. Together, our results suggest that a functional antagonism between the Notch pathway and Ikaros controls normal megakaryocyte development and that this axis is deregulated in AMKL, contributing to aberrant expansion of immature megakaryoblasts. Disclosures: No relevant conflicts of interest to declare.

PU.1 is an Ets-family transcription factor that is critical for normal hematopoiesis. Knock-out studies of PU.1 demonstrate that it is required for the development of myeloid and lymphoid cells. PU.1 is expressed at graded levels in the immune system, with higher expression in myeloid lineage cells. Previous research has suggested that differences in PU.1 concentration are involved in hematopoietic cell fate decisions. However, the mechanism by which target genes in developing cells sense and respond to different PU.1 concentrations is unknown. To address this question, we inserted a beta-lactamase reporter gene and a neomycin resistance cassette into the first coding exon of PU.1. Mice homozygous for the targeted allele (PU.1BN), and an allele in which neomycin was removed (PU.1BLAC), had graded reductions in PU.1 expression and function. PU.1BN/BN mice display a substantial reduction in fetal and neonatal myelopoiesis and are macrophage deficient, resulting in a failure of vascular regression in the developing eye. A fraction of these mice survive until weaning and have an accumulation of c-Kit-expressing immature myeloid cells in the spleen and bone marrow. In addition, these mice acquire a myelomonocytic infiltration in the retina and hyaloid vessels of the eye. Mice homozygous for the second allele (PU.1BLAC/BLAC) survive less than one week and have a severe reduction in PU.1 activity and a block to myeloid development. Fetal livers from both types of mice lack any discernable mature myeloid cells and demonstrate a graded reduction in colony forming ability. To determine if these two alleles have an effect on gene expression, we established IL-7-dependent pro-B cell lines or IL-3-dependent cell lines from these mice as well as from wild type or PU.1 null controls. We found that PU.1-dependent target genes in these cell lines are expressed in a graded manner. In particular, expression of the gene encoding the low-affinity IgG Fc receptor (FcγRIIb) is directly responsive to PU.1 activity. In summary, these results demonstrate the critical importance of an appropriate threshold of PU.1 activity for normal hematopoiesis. The hypomorphic PU.1 alleles provide an excellent model for determining the mechanism of graded PU.1 levels in hematopoiesis.