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The cellular mechanisms required to ensure homeostasis of the hematopoietic niche and the ability of this niche to support hematopoiesis upon stress remain elusive. We here identify Wnt5a in Osterix+ mesenchymal progenitor and stem cells (MSPCs) as a critical factor for niche-dependent hematopoiesis. Mice lacking Wnt5a in MSPCs suffer from stress-related bone marrow (BM) failure and increased mortality. Niche cells devoid of Wnt5a show defective actin stress fiber orientation due to an elevated activity of the small GTPase CDC42. This results in incorrect positioning of autophagosomes and lysosomes, thus reducing autophagy and increasing oxidative stress. In MSPCs from patients from BM failure states which share features of peripheral cytopenia and hypocellular BM, we find similar defects in actin stress fiber orientation, reduced and incorrect colocalization of autophagosomes and lysosomes, and CDC42 activation. Strikingly, a short pharmacological intervention to attenuate elevated CDC42 activation in vivo in mice prevents defective actin-anchored autophagy in MSPCs, salvages hematopoiesis and protects against lethal cytopenia upon stress. In summary, our study identifies Wnt5a as a restriction factor for niche homeostasis by affecting CDC42-regulated actin stress-fiber orientation and autophagy upon stress. Our data further imply a critical role for autophagy in MSPCs for adequate support of hematopoiesis by the niche upon stress and in human diseases characterized by peripheral cytopenias and hypocellular BM.

Hematopoietic stem cell self-renewal, proliferation, and differentiation are independently regulated by intrinsic as well as extrinsic mechanisms. We previously demonstrated that murine proliferation of hematopoietic stem cells is supported in serum-free medium supplemented with two growth factors, stem cell factor and interleukin 11. The survival of hematopoietic stem cells is additionally improved by supplementing this medium with two more growth factors, neural growth factor and collagen 1 (four growth factors) or serum-free medium conditioned by the hematopoietic stem cell-supportive stromal UG26-1B6 cells1. Here, we describe a robust and versatile alternative source of conditioned medium from mouse embryonic fibroblasts. We found that this conditioned medium supports survival and phenotypical identity of hematopoietic stem cells, as well as cell cycle entry in single cell cultures of CD34- CD48- CD150+ Lineage- SCA1+ KIT+ cells supplemented with two growth factors. Strikingly, in comparison with cultures in serum-free medium with four growth factors, conditioned medium from mouse embryonic fibroblasts increases the numbers of proliferating clones and the number of Lineage- SCA1+ KIT+ cells, both with two and four growth factors. In addition, conditioned medium from mouse embryonic fibroblasts supports self-renewal in culture of cells with short- and long-term hematopoiesis-repopulating ability in vivo. These findings identify conditioned medium from mouse embryonic fibroblasts as a robust alternative serumfree source of factors to maintain self-renewal of in vivo-repopulating hematopoetic stem cells in culture.

The support of hematopoiesis declines with age and it was previously shown that aging hematopoietic cells show cytoskeletal deregulation. Also, efficient autophagy is required to maintain cellular functions with age. Here, we describe a new mouse model with reduced support of hematopoiesis and age-related cytoskeletal changes in which the Wnt5a gene is deleted in Osterix (SP7)+ niche cells (O5AD/D mice). Considering that in these mice Wnt5a is deleted in niche cells, we here investigate niche cells from O5AD/D and control mice treated with 5-fluorouracil (5FU). We found that multipotent stromal cells (MSC) from the bone marrow of 5FU-treated O5AD/D mice show reduced CFU-F frequencies with preferential adipogenic differentiation. In these cells, the F-actin network is disorganized and associated with an elevated activity of the actin assembly-stimulating CDC42. Moreover, we found more and perinuclear accumulated ATG7+ and LC3+ autophagosomes in O5AD/D MSC. Also, LAMP1 expression was elevated and lysosomal diameters increased. But, we found no colocalization of LC3 and LAMP1 in O5AD/D MSC and concomitant autophagic flux, suggesting a defect in autophagy. Since in deregulated MSC from 5FU-treated O5AD/D mice CDC42 activity was increased and protein location was different, we additionally treated these mice with the CDC42 inhibitor CASIN. This treatment restored the CDC42 localization in O5AD/D MSCs and their F-actin stress fibers. Furthermore, LC3 and LAMP1 colocalization, mitochondrial and lysosomal diameters, and autophagic flux were restored. Most importantly, CASIN treatment restored the presence of the hematopoietic system and rescued the ability of O5AD/D mice to survive serial 5FU treatments.

We have previously found that a Sfrp1 knock-out environment fails to support the regeneration of hematopoetic stem cells (HSC) with serial to repopulate secondary recipients Renstrom et al., [1] . In order to dissect cell specific requirements of the Sfrp1 gene we established the Sfrp1flox/flox mouse strain and deleted Sfrp1 gene in Osterix+ (Sp7) osteolineage cells. In these Osx-Cre, Sfrp1 (OS1) mice, the number of MSCs is reduced, but these show an increased proportion of colony forming units (CFU-F). Furthermore, stromal cells grown ex-vivo show increased senescence-associated beta-galactosidase staining. In addition, the CFU-F-derived stromal cells differentiated spontaneously into adipocytes. These findings indicate altered functionality of stromal cells from OS1 mice. In the hematopoietic compartment of these mice, we found a decrease in myeloid progenitors in the bone marrow (BM) with concomitant increase in CD11b+ GR1hi granulocytes in peripheral blood. Although the number of primitive CD34- CD48- CD150+ HSCs (LT-HSCs) in the BM was unchanged, LT-HSCs from OS1 mice failed to repopulate in wild type recipients. In single cell cultures, we found that LT-HSCs from OS1 mice show decreased proliferation with concomitant increased differentiation into mature myeloid cells, which was associated with increased DNA damage as shown with comet tail assays and staining for gammaH2.AX. On a molecular level, we found that LT-HSCs from OS1 mice show increased CTNNB1 protein levels. CTNNB1 regulates cell differentiation and proliferation of stem cells by binding to Ep300 or CBP respectively Miyabayashi et al., [2] . Interestingly, CBP protein level was decreased in LT-HSCs from OS1 mice while Ep300 was increased, suggesting overactivation of the CTNNB1/Ep300 axis. Indeed, blocking CTNNB1/Ep300 binding with specific PP2A inhibitor IQ-1, we not only rescued the aberrant behavior of OS1 LT-HSC in vitro, but we also restored the repopulating activity of these LT-HSCs in vivo. Our results suggest that deletion of stromal SFRP1 diminishes the repopulating activity of LT-HSCs by increasing differentiation through PP2A-mediated dephosphorylation of the phospho-Ep300-binding site with CTNNB1.

Successful tissue regeneration depends on the regenerative potential of the graft and its integration in recipients tissue. In bone marrow transplantation (BMT), age-related factors negatively impact on BMT. To dissect the separate contributions of recipient conditioning and aging for the success of BMT, we compared in vitro behavior and transcriptomes of multipotent stromal cells (MSCs) from young (Y: 3 months old), middle aged mice (A: 13 months old) with mice 10 months after BMT (BMT; total age of 13 months). Our experiments show that although HSC numbers are similar in A, and BMT mice, repopulating activity is significantly reduced in BMT HSC. This is accompanied by a reduction of (CD45/Ter119) - CD31 - CD166 - SCA1 + MSCs in the BM with a strongly reduced CFU-F frequency, indicating a functional compromise of BMT MSC. On the cellular level, BMT MSC show a reduced number of mitochondria with increased ROS production compared to A mice. In addition, BMT MSC show disorganized F-actin stress fibers compared to both Y and A MSC. To understand the mechanisms underlying the compromised function of BMT MSC, we analyzed the transcriptome of uncultured primary MSCs from the BM of Y, A, and BMT mice. These analyses show that in comparison to Y and A MSC, BMT MSC downregulate genes involved in intracellular nutrient transport and mitochondrial clearance, but upregulate metabolic processes, lysosomal genes and calcium-dependent non-canonical Wnt signaling. In experiments to assess similar changes in human BMT MSC, we found that MSC from patients undergoing BMT (2 and 4 weeks after BMT) show a severe dysregulation of F-Actin organization, compared to MSC from young and aged healthy donors (HD). Our results show deregulation of HSCs and MSCs in murine or human recipients undergoing BMT. Our results further suggest underlying defects in mitochondrial clearance and metabolic activation with deregulated F-actin organization. Our findings help to dissect and understand mechanisms of age-related factors hampering BMT and could help in devising strategies to improve long-term restoration of tissues after transplantation in aging graft recipients.

Summary Hematopoietic stem cells (HSCs) are preserved in co-cultures with UG26-1B6 stromal cells or their conditioned medium. We performed a genome-wide study of gene expression changes of UG26-1B6 stromal cells in contact with Lineage− SCA-1+ KIT+ (LSK) cells. This analysis identified connective tissue growth factor (CTGF) to be upregulated in response to LSK cells. We found that co-culture of HSCs on CTGF knockdown stroma (shCtgf) shows impaired engraftment and long-term quality. Further experiments demonstrated that CD34− CD48− CD150+ LSK (CD34− SLAM) cell numbers from shCtgf co-cultures increase in G0 and senescence and show delayed time to first cell division. To understand this observation, a CTGF signaling network model was assembled, which was experimentally validated. In co-culture experiments of CD34− SLAM cells with shCtgf stromal cells, we found that SMAD2/3-dependent signaling was activated, with increasing p27Kip1 expression and downregulating cyclin D1. Our data support the view that LSK cells modulate gene expression in the niche to maintain repopulating HSC activity., Graphical Abstract, Highlights • LSK cells induce the expression of CTGF in stromal cells • Stromal CTGF drives cell-cycle progression and proliferation of CD34− LSK cells • Decreased stromal CTGF activates TGFB signaling to decrease LSK cell cycle • Stromal CTGF improves maintenance of HSCs with long-term repopulating activity, Oostendorp and colleagues show that hematopoietic stem cells (HSCs) induce expression of connective tissue growth factor (CTGF) in stromal cells. A decrease in stromal CTGF impairs the maintenance of HSC activity in co-culture by rapid modulation of HSC signaling and cell-cycle entry. Thus, HSCs regulate their own cell cycle by modulating gene expression in stromal cells.

Cyclin-dependent kinase subunit 1 (Cks1) is a critical rate-limiting component of the Skp1-Cullin1-Skp2 (SCF(Skp2)) ubiquitin ligase that controls cell cycle inhibitor abundance. Cyclin-dependent kinase (Cdk) inhibitors (CKIs) regulate hematopoietic stem cell (HSC) self-renewal, regeneration after cytotoxic stress and tumor cell proliferation. We thus studied the role of Cks1 in HSC and in a prototypic stem cell disorder, chronic myeloid leukemia (CML). Cks1 transcript was highly expressed in Lin-Sca-1+Kit+ (LSK) HSC, and the loss resulted in accumulation of the SCF(Skp2)/Cks1 substrates p21, p27, p57 and p130 particularly in CD150+ LSK cells. This accumulation correlated with decreased proliferation and accumulation of Cks1(-/-) HSC, slower regeneration after stress and prolonged HSC quiescence. At the hematopoietic progenitor (HPC) level, loss of Cks1 sensitized towards apoptosis. In CML, Cks1 expression was increased, and treatment with the Abl kinase inhibitor, imatinib, reduced Cks1 expression. Also, we found that Cks1 is critical for Bcr-Abl-induced cytokine-independent clonogenic activity. In conclusion, our study presents a novel function of Cks1 in maintaining HSC/HPC homeostasis and shows that Cks1 is a possible target in therapies aimed at the SCF(Skp2)/Cks1 complex that controls CKI abundance and cancer cell proliferation.

Hematopoietic stem cells (HSCs) are a rare population of somatic stem cells that have the ability to regenerate the entire mature blood system in a hierarchical way for the duration of an adult life. Adult HSCs reside in the bone marrow niche. Different niche cell types and molecules regulate the balance of HSC dormancy and activation as well as HSC behavior in both normal and malignant hematopoiesis. Here, we describe the interplay of HSCs and their niche, in particular the involvement of the Wnt signaling pathway. Although the prevailing notion has been that malignant transformation of HSCs is the main cause of leukemia, evidence is mounting that disruption of niche regulation by transformed hematopoietic cells, which may overexpress Wnt signaling or intrinsic stromal defects in gene expression, is at least a collaborative factor in leukemogenesis. Thus, insights into the normal and altered functions of niche components will help to obtain a better understanding of normal and malignant hematopoiesis and how environmental factors affect these processes.

Inherited bone marrow failure syndromes (IBMFS) are a heterogeneous group of disorders characterized by impaired stem cell function resulting in pancytopenia. Diagnosis of IBMFS presents a major challenge due to limited diagnostic tests and overlapping phenotypes. For that reason, novel and clinical relevant biomarkers and possible targets are urgently needed. Our study defines NIPA as an IBMFS gene, which is significantly downregulated in a distinct subset of MDS-type refractory cytopenia of childhood patients. Mechanistically, NIPA binds FANCD2 and regulates its nuclear abundance. The stabilization of both non- and monoubiquitinated FANCD2 identifies NIPA as an essential player in the Fanconi Anemia (FA) pathway. NIPA thereby prevents MMC hypersensitivity visualized by increased numbers of chromosome radials and reduced cell survival after induction of interstrand crosslinks. To provide proof of principle, re-expression of FANCD2 in Nipa deficient cells restores MMC sensitivity. In a knockout mouse model, Nipa deficiency leads to major cell intrinsic long-term repopulation defects of hematopoietic stem cells (HSCs), with impaired self-renewal in serial transplantations and a bias towards myeloid differentiation. Unresolved DNA damage in Nipa deficient HSCs causes increased sensitivity to cell death and leads to progressive, age-related loss of the HSC pool. Induction of replication stress triggers the phenotypic reduction and functional decline of murine HSCs, resulting in complete bone marrow failure and death of the mice thereby mimicing Fanconi Anemia. Taken together, our study adds NIPA to the short list of FA-associated proteins being essential for a functional DNA repair/FA/BRCA axis and thereby emphasizing its impact as potential diagnostic marker and/or possible target in bone marrow failure syndromes. Disclosures Niemeyer: Celgene: Consultancy.

GATA2 Zinc-Finger (ZF) mutations are associated with specific subgroups of myeloid malignancies. Alterations of the N-terminal ZF1 were identified in AML patients with biallelic CEBPA mutations, whereas the C-terminal ZF2 is typically affected by germline mutations, predisposing to MDS and AML, or by somatic lesions in CML blast crisis. Nevertheless, the context-dependent mechanism underlying GATA2 ZF2 mutations remains mostly unclear. Here, we set out to study the functional consequences of GATA2 ZF mutations in myeloid malignancies. In particular, we performed DNA-pulldown experiments, using FLAG-tagged full length GATA2 ZF WT and mutant proteins after expression in HEK293T cells and incubation of the cell lysates with biotinylated oligonucleotides encoding the binding-motif GATC. These experiments showed disruption of DNA binding for all GATA2 ZF mutants tested in vitro - regardless of the mutant positions within the ZF domains (Figure 1 A). Moreover, we studied the impact of GATA2 ZF mutations on the protein-interaction with Friend of GATA Protein 1 (FOG1; HGNC symbol ZFPM1). The influence of FOG1 on GATA2-dependent transcriptional activation was evaluated using a GATA-specific luciferase reporter. All GATA2 mutants tested were able to activate this reporter, although to variable extent. While co-expression of FOG1 overall counteracted GATA2-dependent transcriptional activation, this effect was significantly reduced for the GATA2 mutants L321F (located in ZF1) and T354M (located in ZF2). This suggested that both ZF domains are involved in the FOG1-interaction. To gain insights into the influence of GATA2 ZF1 mutation on hematopoiesis we performed colony forming cell (CFC)-assays. Lin- primary murine bone marrow cells expressing GATA2 WT or mutants were plated in methylcellulose supplemented with cytokines. Consistent with previous reports, GATA2 WT expression led to a reduced colony number, while this effect was decreased for both mutants A318T (ZF1) and L359V (ZF2). In particular, a higher number of CFU-G colonies were observed for the GATA2 mutant-expressing cells indicating a lineage shift towards granulopoiesis (Figure 1 B). In summary, we have shown that GATA2 mutations influence DNA-binding, protein-interactions and myeloid differentiation. Our findings further suggest that GATA2 ZF1 mutations may contribute to myeloid leukemogenesis through increased proliferation of granulocytic progenitors. Understanding the oncogenic collaboration of GATA2 mutations with other driver genes in distinct patient subgroups is a challenge ahead. Disclosures Hiddemann: Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; F. Hoffman-La Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Consultancy, Research Funding.

Sfrp2 is overexpressed in stromal cells which maintain hematopoietic stem cells (HSCs) during in vitro culture. We here showed, that coculture of hematopoetic cells with stromal cells with reduced expression of Sfrp2 increases the number lineage-negative Kit+ Sca-1+ (LSK) and progenitor cells in vitro. The LSK cells from these cocultures showed activation of canonical Wnt signaling, higher levels of Ki-67, BrdU incorporation, and the number of γH2A.X positive foci. Total repopulating activity of these cultures was, however, diminished, indicating loss of HSC. To extend these in vitro data, we modelled stress in vivo, i.e., by aging, or 5-FU treatment in Sfrp2−/− mice, or replicative stress in regeneration of HSCs in Sfrp2−/− recipients. In all three in vivo stress situations, we noted an increase of LSK cells, characterized by increased levels of β-catenin and cyclin D1. In the transplantation experiments, the increase in LSK cells in primary recipients was subsequently associated with a progressive loss of HSCs in serial transplantations. Similar to the in vitro coculture stress, in vivo genotoxic stress in 5-FU-treated Sfrp2−/− mice increased cell cycle activity of LSK cells with higher levels of BrdU incorporation, increased expression of Ki-67, and canonical Wnt signaling. Importantly, as noted in vitro, increased cycling of LSKs in vivo was accompanied by a defective γH2A.X-dependent DNA damage response and depolarized localization of acetylated H4K16. Our experiments support the view that Sfrp2 expression in the niche is required to maintain the HSC pool by limiting stress-induced DNA damage and attenuating canonical Wnt-mediated HSC activation.

Pleiotrophin (Ptn) is strongly expressed by stromal cells which maintain HSCs. However, in vivo, Ptn deficiency does not alter steady-state hematopoiesis. However, knockdown of Ptn (PtnKD) in stromal cells increases production of hematopoietic progenitors as well as HSC activity in cocultures, suggesting that Ptn may have a role in HSC activation. Indeed, transplantations of wild-type (Ptn+/+) HSCs into Ptn−/− mice show increased donor cell production in serial transplantations and dominant myeloid regeneration caused by Ptn-dependent regulation of HSC repopulation behavior. This regulation of Lin−Kit+Sca1+ function is associated with increased proliferation and, on a molecular level, with up-regulated expression of cyclin D1 (Ccnd1) and C/EBPα (Cepba), but reduced of PPARγ. The known HSC regulator β-catenin is, however, not altered in the absence of Ptn. In conclusion, our results point to different Ptn-mediated regulatory mechanisms in normal hemostasis and in hematopoietic regeneration and in maintaining the balance of myeloid and lymphoid regeneration. Moreover, our results support the idea that microenvironmental Ptn regulates hematopoietic regeneration through β-catenin–independent regulation of Ccnd1 and Cebpa.

Summary Secreted frizzled-related protein 1 ( Sfrp1 ) is highly expressed by stromal cells maintaining hematopoietic stem cells (HSCs). Sfrp1 loss in stromal cells increases production of hematopoietic progenitors, and in knockout mice, dysregulates hemostasis and increases Flk2− Cd34− Lin− Sca1+ Kit+ (LSK) cell numbers in bone marrow. Also, LSK and multipotent progenitors (MPPs) resided mainly in the G0/G1 phase of cell cycle, with an accompanying decrease in intracellular β-catenin levels. Gene-expression studies showed a concomitant decrease Ccnd1 and Dkk1 in Cd34− LSK cells and increased expression of Pparg , Hes1 , and Runx1 in MPP. Transplantation experiments showed no intrinsic effect of Sfrp1 loss on the number of HSCs or their ability to engraft irradiated recipients. In contrast, serial transplantations of wild-type HSCs into Sfrp1 −/− mice show a progressive decrease of wild-type LSK and MPP numbers. Our results demonstrate that Sfrp1 is required to maintain HSC homeostasis through extrinsic regulation of β-catenin.

Accumulation of DNA damage in hematopoietic stem cells (HSCs) is associated with aging, bone marrow failure and development of hematological malignancies. Although HSCs numerically expand with age, their functional activity declines over time and the protection mechanism from DNA damage accumulation remains to be elucidated. NIPA (Nuclear Interaction Partner of ALK) is highly expressed in hematopoietic stem and progenitor cells, especially in the most primitive long-term repopulating HSCs (CD34-Flt3-Lin-Sca1+cKit+). Loss of NIPA leads to a significant exhaustion of primitive hematopoietic cells, where Lin-Sca1+cKit+ (LSK) cells were reduced to 40% of wildtype (wt) littermates (p To further examine the role of NIPA in HSC maintenance and exhaustion, we performed in vivo repopulationexperiments, where Nipa deletion causes bone marrow failure in case of competition, as Nipako/ko cells contributed to less than 10% of transplanted BM cells 6 month after transplantation (TX). According to this, colony formation assays and limiting dilution transplantation showed a dramatic reduction of competitive repopulation units (p To further investigate the stress response in Nipa-deficient HSCs, we irradiated LSKs with 3 Gy and stained for DNA-Damage foci by pH2ax. Remarkably, loss of NIPA led to significant higher numbers of pH2ax foci in irradiated HSCs (46% > 6 foci vs. 17% > 6 foci in wt cells) and highly increased the rates of apoptotic cells especially in the primitive CD34-LSK population. Taken together our results highlight the importance of the DNA damage response at HSC level for lifelong hematopoiesis and establish NIPA as a novel regulator of aging and stress response of the primitive HSC pool. Disclosures No relevant conflicts of interest to declare.

Recently, mutations of additional sex comb-like 1 (ASXL1) have been identified as an independent poor prognostic factor in MDS patients. We previously demonstrated that C-terminal–truncating ASXL1 mutations (ASXL1-MT) inhibited myeloid differentiation and induced MDS-like disease in mice by inhibiting polycomb repressive complex 2–mediated methylation of histone H3K27. Intriguingly, the patients with ASXL1 mutation have significantly higher incidences of concurrent SET binding protein 1 (SETBP1) mutation than those with wild-type ASXL1, which prompted us to investigate whether SETBP1 mutation plays a critical role in leukemic transformation of MDS. Most SETBP1 mutations, such as D868N, have been shown to occur in the PEST domain of ski homology region, resulting in the prevention from proteasomal degradation. In in vitro experiment, the expression of both SETBP1-D868N and ASXL1-MT enhanced myeloid colony output, reinforced ASXL1-MT-induced differentiation block and inhibited apoptosis. Of note, SETBP1-D868N collaborated with ASXL1-MT in inducing MDS/AML in mice BMT model compared to control mice (0.0001). Using GSEA, we demonstrated that SETBP1-MT enriched hematopoietic stem cells-related genes and deregulated p53 and Myc pathway. In addition, SETBP1-MT inhibited PP2a leading to the activation of the Akt pathway and exhibited characteristic histone modification pattern. In conclusion, our data provide evidence for the role of SETBP1-MT in leukemic transformation and suggest the deregulated pathways as a potential therapeutic target in MDS to prevent disease progression.

Hematopoietic stem cells (HSC) are regulated by an interplay of intrinsic and extrinsic signals, the latter of which are mostly transmitted by the niche. The processes involved and their interactions are largely unknown. We studied the dynamic interaction of HSC and niche stromal cells, using co-cultures of HSC (lineage-negative Sca-1+ c-Kit+: LSK) cells and HSC-maintaining UG26-1B6 stromal cells. Microarray analyses from cells prior to co-culture and cells sorted separately from the cultures revealed that most changes in gene expression take place in the first 24 hours of co-culture. Analyses using STEM clustering, LIMMA, and ToppGene databases showed early activation of cell cycle progression In LSK cells and extensive remodeling of chomatin structure and transcriptional activation in both LSK and stromal cells. Interestingly, connective tissue growth factor (Ctgf/Ccn2), which is involved in TGFb, BMP and Wnt signaling, was strongly upregulated in both stromal and LSK cells. To study the role of Ctgf as a stromal mediator, LSK cells were co-cultured with siCTGF knockdown stromal cells. We showed that although short-term HSC activity was unchanged, siCtgf-stromal cells were unable to sustain long-term repopulating ability. To study underlying mechanisms, a Boolean model simulating possible signaling mechanisms leading to cell cycle activation was extracted from the data. We validated this model by co-cultures of LSK cells with control and Ctgf-knockdown stroma, separating LSK and stromal cells and assessing protein levels and phosphorylation using immunocytofluorescence in LSK cells. We found that in the absence of extrinsic Ctgf, expression of Pten was increased in LSK cells. However, phosphorylation of Akt (both p308, and p473) and Erk was unchanged. In contrast, both canonical Wnt (LRP6, Gsk3b, b-catenin) and Tgfb (Smad2/3) signaling were significantly affected in that Wnt signaling was turned off, whereas Tgf signaling was turned on, by the lack of extrinsic Ctgf. This resulted in a downregulation of G1 transition, as was exemplified by downregulation of Cyclin D1, upregulation of p27Kip1 and modulations in the phosphoryalation of both Rb and p53. Equally interestingly, we could show that extrinsic Ctgf deficiency also downregulates induction of Ctgf in HSC, suggesting the existence of intrinsic-extrinsic feedback signaling. In summary, co-culture of LSK cells with stromal cells results in cellular activation of both stromal cells and LSK cells, involving Tgf and canonical Wnt signaling pathways. Furthermore, reduced expression of extrinsic Ctgf, regulates mediators of G1 cell cycle progression in LSK cells an a biochemical level and functionally results in an increased production of myeloid progenitors and decreased long-term repopulating ability. Our studies show the dynamics of reciprocal signaling between HSC and niche stromal cells and give insights how the niche regulates early regenerative responses in hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

Abstract 771 Introduction: Activating mutations of FLT3 are among the most frequent alterations in AML. Two distinct types of mutations have been described: Duplication of the juxtamembraneous domain (ITD) and point mutations of the tyrosine kinase domain (TKD). Although both lead to constitutive FLT3 signaling, FLT3-ITD signals through the STAT5 pathway whereas FLT3-TKD and FLT3 WT activate STAT5 to a much lesser degree. In a murine bone marrow transplantation model, FLT3-ITD leads to a myeloproliferative neoplasm; FLT3-TKD induces a lymphoid disease with significantly longer latency. Interaction of SRC with FLT3-ITD was recently shown to be necessary for STAT5 activation. Methods: Fetal liver cells from constitutive STAT5 knockout mice were infected with FLT3 oncogenes and cultured on methylcellulose. Conditional STAT5 knockout mice were analyzed for phenotype and latency of hematologic disorders after transplantation with FLT3-ITD or FLT3-TKD-infected bone marrow. Expression of STAT5 target genes was determined using Realtime-PCR. Phospho-STAT5 levels were quantified by intracellular staining and flow cytometry. Results: Depletion of STAT5 leads to a dramatic decrease in the ability of fetal liver cells to form colonies in Methylcellulose when infected with FLT3-ITD, but not when infected with FLT3-TKD. In-vivo studies using a murine conditional knockout model proved that STAT5 is necessary for the induction of a myeloproliferative disease by FLT3-ITD, while FLT3-TKD is not dependent on STAT5. Mice with deletion of the STAT5 allele displayed expansion of mainly lymphoid cells with an increased latency similar to the FLT3-TKD disease. In additional studies differential activation of STAT5 in myeloid and lymphoid progenitors was found. The STAT5 target gene Oncostatin M is exclusively expressed in myeloid progenitor cells infected with FLT3-ITD and in mice with intact STAT5 that were transplanted with FLT3-ITD. Furthermore, elevated SRC expression levels could be detected in proliferating myeloid progenitor cells compared to lymphoid progenitors. Conclusion: STAT5 is critical for the induction of a myeloproliferative disease by FLT3-ITD in a murine transplantation model. Interaction of SRC and FLT3-ITD leads to the activation of STAT5 in myeloid progenitors, but not in lymphoid progenitors expressing low levels of SRC. Oncostatin M upregulation is likely to be a downstream process contributing to the phenotype and latency of the disease. Disclosures: No relevant conflicts of interest to declare.

Abstract 2353 Hematopoiesis is maintained throughout life by the constant production of mature blood cells from hematopoietic stem cells (HSC). One mechanism by which the number of HSC is maintained is self-renewal, a cell division in which at least one of the daughter cells is a cell with the same functional potential as the mother cell. The mechanisms of this process are largely unknown. We have described cell lines that maintain self-renewal in culture. To study possible mechanisms and mediators involved in self-renewal, we performed co-cultures of HSC model cells: Lineage-negative Sca-1+ c-Kit+ (LSK) cells and HSC maintaining UG26–1B6 stromal cells. Microarray analyses were performed on cells prior to co-culture and cells sorted from the cultures. STEM clustering analysis of the data revealed that most changes in gene expression were due to early cell activation. Functional enrichment analysis revealed dynamic changes in focal adhesion and mTOR signaling, as well as changes in epigenetic regulators, such as HDAC in stromal cells. In LSK cells, genes whose products are involved in inflammation, Oxygen homeostasis and metabolism were differentially expressed after the co-culture. In addition, genes involved in the regulaton of H3K27 methylation were also affected. Interestingly, connective tissue growth factor (CTGF), which is involved in TGF-b, BMP and Wnt signaling, was upregulated in both stromal and LSK cells in the first day of co-culture. To study a possible extrinsic role of CTGF as a stromal mediator, we co-cultured siCTGF knockdown stromal cells with wild-type LSK cells. Since self-renewal requires cell division, we focused on cell cycle regulation of LSK cells. We found that knockdown of CTGF in stromal cells downregulates CTGF in LSK cells. In addition, knockdown of stromal CTGF downregulated Ccnd1, Cdk2, Cdkn1a (p21), Ep300 and Fos. On the other hand, decreased CTGF in stromal cells upregulates Cdkn1b (p27) and phosphorylation of Smad2/3. These results show that stromal CTGF regulates the cell cycle of LSK cells. On a functional level, we found that decreased stromal CTGF results in an increased production of MPP and myeloid colony-forming cells in 1-week co-cultures. We will present data showing whether and how a decrease in CTGF in stromal cells affects the maintenance of transplantable HSC. In summary, our current results indicate that reduced expression of CTGF in stromal cells regulates mediators of cell cycle and Smad2/3-mediated signaling in LSK cells, resulting in an increased production of myeloid progenitors. Disclosures: No relevant conflicts of interest to declare.

Abstract 2330 Hematopoietic stem cells (HSCs) are characterized by their ability to self-renewal and multilineage differentiation. Since mostly HSCs exist in a quiescent state re-entry into cell cycle is essential for their regeneration and differentiation and the expression of numerous cell cycle regulators must be tightly controlled. We previously characterized NIPA (Nuclear Interaction Partner of ALK) as a F-Box protein that defines an oscillating ubiquitin E3 ligase targeting nuclear cyclin B1 in interphase thus contributing to the timing of mitotic entry. To examine the function of NIPA on vivo, we generated NIPA deficient animals, which are viable but sterile due to a defect in recombination and testis stem cell maintenance. To further characterize the role of NIPA in stem cell maintenance and self-renewal we investigated hematopoiesis in NIPA deficient animals. Peripheral blood counts taken at different ages revealed no apparent difference between NIPA knockout and wild type mice in numbers and differentiation. In contrast, looking at the hematopoietic stem cell pool, FACS analyses of bone marrow showed significantly decreased numbers of Lin-Sca1+cKit+ (LSK) cells in NIPA deficient animals, where LSKs were reduced to 40% of wild type littermates (p=0,0171). This effect was only apparent in older animals, where physiologically higher LSK numbers have to compensate for the exhaustion of the stem cell pool. Additionally, older NIPA deficient mice have only half the amount of multi myeloid progenitors (MMPs) in contrast to wild type animals. To examine efficient activation of stem cells to self-renew in response to myeloid depression, we treated young and old mice with the cytotoxic drug (5-FU) four days before bone marrow harvest. As expected, 5-FU activated hematopoietic progenitors in wild type animals, whereas NIPA deficient progenitors failed to compensate to 5-FU depression, e.g. LSKs of NIPA knockout mice were reduced to 50% of wild type levels (p Taken together our results demonstrate a unique and critical role of NIPA in regulating the primitive hematopoietic compartment as a regulator of self-renewal, cycle capacity and HSC expansion. Disclosures: No relevant conflicts of interest to declare.