ALBERT

Description: Key Points C/EBPα is needed for transition from stem/progenitor cells to common dendritic cell progenitors. C/EBPα is dispensable in later stages of dendritic cell maturation.

Description: Recent evidence suggests that mutations in the Gata1 gene may alter the proliferation/differentiation potential of hemopoietic progenitors. By single-cell cloning and sequential replating experiments of prospectively isolated progenitor cells, we demonstrate here that the hypomorphic Gata1low mutation increases the proliferation potential of a unique class of progenitor cells, similar in phenotype to adult common erythroid/megakaryocytic progenitors (MEPs), but with the “unique” capacity to generate erythroblasts, megakaryocytes, and mast cells in vitro. Conversely, progenitor cells phenotypically similar to mast cell progenitors (MCPs) are not detectable in the marrow from these mutants. At the single-cell level, about 11% of Gata1low progenitor cells, including MEPs, generate cells that will continue to proliferate in cultures for up to 4 months. In agreement with these results, trilineage (erythroid, megakaryocytic, and mastocytic) cell lines are consistently isolated from bone marrow and spleen cells of Gata1low mice. These results confirm the crucial role played by Gata1 in hematopoietic commitment and identify, as a new target for the Gata1 action, the restriction point at which common myeloid progenitors become either MEPs or MCPs.

Description: Recent evidence suggests that mutations in the Gata1 gene may alter the proliferation/differentiation potential of hemopoietic progenitors. We had previously described that hematopoietic tissues from mice carrying the hypomorphic Gata1low mutation contain high numbers (~10%) of “unique” progenitor cells that generate colonies composed by erythroblasts, megakaryocytes and mast cells (Migliaccio et al, J Exp Med197:281, 2003). Predicted by the stochastic model of hematopoietic commitment, such a tri-lineage progenitor cell has not been isolated prospectively as yet from the marrow of normal mice. The aim of this study was to clarify the role of Gata1 in hematopoietic commitment by identifying the antigenic profile and proliferation potential of the Gata1low progenitors giving rise to tri-lineage colonies. First, we compared the frequency of granulo-monocytic (GMP), megakaryocytic-erythroid (MEP), common myeloid (CMP) and mast-cell restricted (MCP) progenitors in marrow, and spleen, from wild-type and Gata1low littermates. Next, the different populations were isolated, and their differentiation and proliferation potential characterized under conditions of limiting dilution followed by single cell re-cloning. In Gata1low mice, the frequency of cells with the antigenic profile of CMP, MEP, and GMP was normal in marrow and markedly increased in spleen, while cells with the MCP profile were not detectable. However, mutant cells isolated according to the MEP phenotype, had the “unique” property to generate mast cells and their precursors in 7 days of culture, in addition to erythroblasts and megakaryocytes. Furthermore, the progeny of ~11% of mutant MEP could be propagated in culture, as single cells, with 95% efficiency, for up of 4 months. In comparison, the progeny of wild-type MEP became extinguished in 7–14 days. In agreement with these results, tri-lineage (erythroid, megakaryocytic and mastocytic) cell lines were consistently isolated from bone marrow and spleen cells of Gata1low mice. These results indicate that cells with the phenotype corresponding to myeloid, but not those corresponding to mastocytic, progenitors are detectable in tissues from Gata1low mice. However, in these mice, the mast cell generating activity is abnormally acquired by MEP, that, therefore, are similar in phenotype, but not in function, to wild-type MEP. These results confirm the crucial role played by Gata1 in hematopoietic commitment and identify, as a new target for the Gata1 action, the restriction point at which CMP became either MEP or MCP.

Description: Chronic Myeloid Leukemia (CML) is a myeloproliferative disorder characterized by the presence of the Philadelphia chromosome deriving from the genetic translocation t(9;22)(q34;q11.2) that encodes for the BCR-ABL fusion gene. BCR-ABL is a constitutively active tyrosine kinase believed to be the primary genetic event driving CML development (Daley and Baltimore, 1988; Huettner et al., 2000). Tyrosine Kinase Inhibitors (TKI) targeting the BCR-ABL kinase have revolutionized CML therapy, however, they fail to fully eradicate the disease due to the presence of a drug-resistant stem cell pool that sustains continued growth of the malignant cells. Indeed, discontinuation of TKI results in relapse and/or disease progression. It has been shown that the emergence of leukemic clones resistant to TKI and responsible for CML evolution is correlated with aberrant DNA methylation (Machova Polakova et al., 2013). DNA methylation is a key epigenetic signature implicated in regulation of gene expression (Robertson, 2001), that occurs predominantly within CpG dinucleotides. CpG-rich regions (namely CpG islands) are frequently located within promoter regions (~70%) of human protein-coding genes (Illingworth et al., 2010). Methylation of CpG-rich promoters negatively correlates with gene expression levels and it is considered an important regulatory mechanism for long-term gene silencing (Herman and Baylin, 2003). Although numerous studies have established a link between aberrant promoter DNA methylation and cancer (Costello et al., 2000; Feinberg et al., 2006), the impact of DNA methylation in CML is still poorly understood (Yamazaki et al., 2012), particularly due to the lack of the specific animal models. In this study we tested the functional relevance of aberrant methylome in CML development and investigated the possibility that BCR-ABL oncogene triggers DNA methylation changes leading to an aggressive leukemic phenotype. To this end, we have combined cellular reprogramming (Amabile and Meissner, 2009; Carette et al., 2010; Kumano et al., 2012; Miyoshi et al., 2010; Takahashi et al., 2007), due to its ability to erase tissue-specific DNA methylation and to re-establish an embryonic stem-like DNA methylation state (Mikkelsen et al., 2008), with a previously developed BCR-ABL inducible murine model (Koschmieder et al., 2005). Using this approach, we demonstrate that the presence of a single genetic aberration is sufficient to trigger DNA methylation changes and leads to an aggressive leukemic phenotype. We show that by resetting the normal DNA methylation profile of primary human CML cells through cellular reprogramming, we are able to re-establish normal myeloid differentiation, despite the persistence of the native genetic lesion. Finally, by combining a comprehensive genome-scale methylation analysis with cellular reprogramming of leukemic cells, we elucidate the sequential events driving leukemogenesis and reveal the reciprocal interplay of genetic and epigenetic mechanisms during malignant transformation. In conclusion, these results dissect the role of DNA methylation alterations in CML development and warrant an application of demethylating agents such as 5-azacytidine as adjuvant treatment in therapeutic approaches to CML. Disclosures Martinelli: Novartis: Speakers Bureau; Bristol Myers Squibb: Speakers Bureau; Pfizer: Speakers Bureau.

Description: Abstract 114 Transcription factor CCAAT/enhancer-binding protein alpha (C/EBPα) controls cell proliferation and myeloid differentiation. In 7∼10% of patients with acute myeloid leukemia (AML) C/EBPA is either mutated or epigenetically silenced. C/EBPA mutated leukemias differ from C/EBPA silenced leukemias in prognosis and phenotype, yet both leukemias cluster together based on genome wide gene expression signatures, indicating a unifying mechanism of disease. So far, the key molecular downstream events required for C/EBPA loss to trigger leukemogenesis are still unclear. Based on microarray gene expression analysis, we here used a shRNA screening platform to search for mediators of leukemic outgrow of C/EBPα-deficient progenitor cells. In our screen, oncogene Sox4 was identified as a gene that was up-regulated in C/EBPα-deficient hematopoietic stem cells (HSCs, both lineage−c-kit+ScaI+ HSCs and SLAM+ HSCs) and whose down-regulation abrogated aberrant self-renewal ability and restored myeloid differentiation of C/EBPα-deficient stem/progenitor cells, as demonstrated by in vitro serial-replating and differentiation assays. Chromatin immunoprecipitation confirmed the endogenous binding of C/EBPα at the proximal promoter of Sox4 in the stem/progenitor cells enriched population (lineage−c-kit+) and the mature myeloid population (Mac1+Gr1+). In vitro promoter reporter assay demonstrated that wild-type human C/EBPA, but none of the C/EBPA mutants identified from AML patients, repressed Sox4 transcription through its binding to a highly conserved C/EBPα binding site. C/EBPα and Sox4 showed reciprocal expression patterns in both HSCs and various hematopoietic compartments during myeloid maturation of wild type mice. Furthermore, expression of Sox4 was up-regulated in HSCs of C/EBPα-deficient mice as well as in leukemia-initiating cells (LICs) of a murine C/EBPα mutant AML model. To further genetically dissect the role of Sox4 in driving leukemia in the absence of functional CEBPα, we generated Sox4, C/EBPα double deficient mice and observed that loss of Sox4 alleviated the abnormal stem/progenitor cell expansion and defective myeloid programming caused by C/EBPα deficiency. In addition, comparisons of the murine C/EBPα mutant AML model with a Sox4-induced AML model revealed that leukemia initiating cells of both leukemia models were enriched in immunophenotypically similar populations and exhibited comparable gene expression signatures. Similar to the C/EBPα knockout model, down-regulation of Sox4 by shRNA in LICs from murine C/EBPα mutant AML was also sufficient to abolish their augmented serial-replating ability. Importantly, enhanced expression of SOX4 in AML patients with either mutated or epigenetically silenced C/EBPA compared to other AML subtypes confirmed the findings in our mouse model systems. Our data demonstrate that failure to suppress Sox4 expression is the underlying mechanism of leukemias with mutated or silenced C/EBPα. These data also uncover a promising rationale for a therapeutic target in these leukemias. Disclosures: No relevant conflicts of interest to declare.

Description: Hematopoietic stem cells are capable of perpetual self-renewal and multi-lineage differentiation, properties that are maintained throughout life by minimal cell cycle activity. Our work has focused on deciphering transcriptional driven differentiation versus self-renewal pathways in stem and progenitor cells. To this end, we have studied transcription factors that control the fate of hematopoietic stem cells by combining mouse models of activated self-renewal with models that can report transcription factor expression. We chose to study the Wnt pathway, activated in several types of leukemia, in combination with the ets family PU.1 transcription factor, vital to almost all myeloid and lymphoid lineages. PU.1 regulates a number of important myeloid specific genes that mediate differentiation to a specific cell fate. To understand the interaction of these pathways, we found that over-expression of Wnt signaling or beta-catenin, the downstream signaling component of the Wnt pathway, was able to inhibit PU.1-mediated differentiation in a PU.1-inducible cell line. There was little to no up-regulation of the myeloid markers Mac1 or Gr1 with activation of Wnt signaling upon induction with 4-hydroxy-tamoxifen (4-OHT). Additionally, many genes related to myeloid differentiation were not increased as compared to control-induced cultures. To understand how these interactions might function in vitro, we crossed a Cre-responsive activated beta-catenin (floxed allele Exon3) mouse to a PU.1-GFP knock-in mouse. From this model, we are able to see changes in PU.1 (GFP) expression in specific populations of hematopoietic progenitors upon activation of beta-catenin. Most importantly, in the LT-HSCs (defined by Lin- cKitHi Sca1+ CD150+ CD48-), we observed a significant increase in GFP (PU.1) intensity upon activation of active beta-catenin. Additionally, there was an increase in the total number of LT-HSCs, as defined by surface markers. LT-HSCs with active beta-catenin and GFP (PU.1) were found to be more in cycle and they express lower levels of transcription factors related to differentiation. These results demonstrate that when beta-catenin is activated, PU.1’s role is modified and the self-renewal program is enhanced at the expense of differentiation. Furthermore, activation of beta-catenin in the hematopoietic cells of mice has been shown to lead to impaired differentiation and eventual death. Even though active beta-catenin has been shown to be essential in several subtypes of myeloid leukemias using murine models, its over-expression is not sufficient to lead to leukemic development. However, heterozygous PU.1/GFP knock-in mice were crossed to the beta-catenin overexpression model, they rapidly developed leukemia post Cre induction. This is not observed in the PU.1/GFP knock-in mice in the absence of beta-catenin activation, suggesting that Wnt signaling adds to a block in differentiation needed for leukemic transformation. These mice show splenomegaly and increased myelocytic populations in the peripheral blood. The leukemia was transplantable to secondary mice and expressed high levels of GFP (PU.1) in the spleen, bone marrow and peripheral blood. These findings demonstrate that the interaction and crosstalk between these two pathways regulate hematopoietic stem cell fate. Future studies will focus on understanding how this interaction between transcription factor and self-renewal pathways becomes disrupted in leukemic stem cells. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 1234 Specialized bone marrow (BM) microenvironment niches are essential for hematopoietic stem and progenitor cell maintenance, and recent publications have focused on the leukemic stem cells interaction and placement within those sites. Surprisingly, little is known about how the integrity of this leukemic niche changes the normal stem and progenitor cells behavior and functionality. To address this issue, we started by studying the kinetics and differentiation of normal hematopoietic stem and progenitor cells in mice with Chronic Myeloid Leukemia (CML). CML accounts for ∼15% of all adult leukemias and is characterized by the BCR-ABL t(9;22) translocation. Therefore, we used a novel SCL-tTA BCR/ABL inducible mouse model of CML-chronic phase to investigate these issues. To this end, BM from leukemic and normal mice were mixed and co-transplanted into hosts. Although normal hematopoiesis was increasingly suppressed during the disease progression, the leukemic microenvironment imposed distinct effects on hematopoietic progenitor cells predisposing them toward the myeloid lineage. Indeed, normal hematopoietic progenitor cells from this leukemic environment demonstrated accelerated proliferation with a lack of lymphoid potential, similar to that of the companion leukemic population. Meanwhile, the leukemic-exposed normal hematopoietic stem cells were kept in a more quiescent state, but remained functional on transplantation with only modest changes in both engraftment and homing. Further analysis of the microenvironment identified several cytokines that were found to be dysregulated in the leukemia and potentially responsible for these bystander responses. We investigated a few of these cytokines and found IL-6 to play a crucial role in the perturbation of normal stem and progenitor cells observed in the leukemic environment. Interestingly, mice treated with anti-IL-6 monoclonal antibody reduced both the myeloid bias and proliferation defects of normal stem and progenitor cells. Results obtained with this mouse model were similarly validated using specimens obtained from CML patients. Co-culture of primary CML patient samples and GFP labeled human CD34+CD38- adult stem cells resulted in selective proliferation of the normal primitive progenitors compared to mixed cultures containing unlabeled normal bone marrow. Proliferation was blocked by adding anti-IL-6 neutralizing antibody to these co-cultures. Therefore, our current study provides definitive support and an underlying crucial mechanism for the hematopoietic perturbation of normal stem and progenitor cells during leukemogenesis. We believe our study to have important implications for cancer prevention and novel therapeutic approach for leukemia patients. We conclude that changes in cytokine levels and in particular those of IL-6 in the CML microenvironment are responsible for altered differentiation and functionality of normal stem cells. Disclosures: No relevant conflicts of interest to declare.