Murine Myeloid Progenitor Cells Research Articles

Interconnections between DDX41-Dependent Post-Transcriptional and GATA Factor-Instigated Transcriptional Mechanisms That Govern Hematopoiesis

Heterozygous DDX41 germline mutations occur in familial myelodysplastic syndrome (MDS)/acute myeloid leukemia (AML) and acute erythroid leukemia (AEL). DDX41 encodes an RNA helicase that regulates RNA splicing, cGAS-Sting signaling in innate immunity, and maintains genome stability in erythropoiesis. We described the innovation of a facile genetic rescue system to compare RNA-regulatory activities of wild type and disease-associated DDX41 variants in engineered HoxB8-immortalized (hi) murine myeloid progenitor cells (hi-Ddx41+/+ and Ddx41+/- cells) (Kim et al., Leukemia 2023). We identified DDX41-regulated transcripts and membrane proteins as quantitative metrics of DDX41 activity that discriminate benign from pathogenic DDX41 alleles. As these transcripts and the cognate proteins were not known to be components of GATA2 genetic networks that govern hematopoietic stem/progenitor cell (HSPC) transitions, we hypothesized that DDX41 mechanisms in progenitors operate in parallel with or independent of GATA2 mechanisms. Considering the biological and pathological importance of DDX41 and many unanswered questions on how clinical variants impact DDX41 activity, we instituted studies to discover DDX41-regulated transcript isoforms. The datasets are being used to unveil how DDX41 controls cellular functions, how clinical variants derail mechanisms, and to establish interconnections or independence with GATA factor mechanisms. To develop a broader perspective, we extended the myeloid data by innovating a rescue system to analyze DDX41 structure/function in G1E-ER-GATA1 erythroblasts that express a conditionally active ER-GATA1 allele. We used CRISPR/Cas9 to generate Ddx41 +/- clonal lines that expressed ~50% lower levels of DDX41 protein. A comparable system is being generated in human HUDEP2 erythroid precursor cells. We compared DDX41 with two disease-associated germline variants: G173R and K331deletion. G1E-ER-GATA1- Ddx41 +/-cells were infected with retrovirus expressing GFP, or GFP and DDX41 (WT, G173R, or K331del), GFP + cells were sorted, and total RNA was analyzed by RNA-seq. Differential expression analysis revealed 138 Ddx41-regulated transcripts (P < 0.05), with 63 elevated and 75 decreased. Disease-associated variants failed to regulate these transcripts. To establish relationships between DDX41-regulated and GATA1-regulated transcripts in erythroblasts, we compared DDX41-regulated transcripts with our GATA1 transcriptomic data (Tanimura et al., EMBO Rep. 2016; Dev. Cell 2018; Liao et al., Cell Rep. 2020), which revealed 78 transcripts co-regulated by DDX41 and GATA1. The RNA analysis is being extended to the proteome. To investigate similarities between DDX41-dependent post-transcriptional and GATA1-dependent transcriptional mechanisms, we analyzed the genes encoding the 78 co-regulated transcripts using gene ontology and pathway analyses. The gene signatures included downregulation of apoptosis (p=0.0006). We are testing the hypothesis that DDX41 and GATA1 operate additively or synergistically to control levels of transcripts encoding apoptotic regulators in developing erythroblasts, thus establishing a balance between pro-apoptotic and anti-apoptotic factors. We compared myeloid and erythroid DDX41-regulated transcript datasets to identify common and cell type-specific DDX41-regulated transcripts. This analysis unveiled 11 transcripts that are DDX41-regulated in both systems. One transcript encodes CLK3, a member of Cdc2-like kinase family that phosphorylates splicing factors. DDX41 increased intron 3 retention in Clk3 mRNA in both systems. By quantifying exon-intron junctions, we demonstrated that DDX41 promoted intron 3 retention in hi- Ddx41 +/- cells 1.7-fold (P = 0.0002) and in G1E-ER-GATA1- Ddx41 +/- cells 3.3-fold (P = 0.0002). We are testing models to explain the functional significance of DDX41-regulated Clk3 intron retention and discovered DDX41 regulates intron retention at other loci. We are assessing how DDX41 controls alternative splicing globally in the erythroid and myeloid systems and elucidating the biological and pathological consequences of corrupting these mechanisms. In aggregate, these studies established a system to discriminate pathogenic from benign DDX41 variants, and mechanistic insights that provide the basis for new myeloid and erythroid paradigms.

LSD1 Inhibition Enhances Antigen Presentation and Co-Stimulation in AML to Promote T Cell-Mediated Anti-Leukemia Immune Responses

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) can provide a curative treatment option for acute myeloid leukemia (AML). Donor T cells play an important role in the curative potential of allo-HSCT by recognizing and eliminating residual leukemic cells, inducing the desirable graft-versus-leukemia (GVL) effect. Evading from this effect of donor T cell immune surveillance through the downregulation of human leukocyte antigen class II (HLA-II) molecules has been shown to contribute to AML relapse after allo-HSCT. Lysine-specific demethylase 1 (LSD1) is an emerging epigenetic therapeutic target in AML due to its role in regulating myeloid differentiation pathways. We hypothesized that LSD1 inhibition has the potential to enhance anti-leukemic immune responses by differentiating AML cells and inducing the acquisition of markers typical for antigen-presenting cells. We have studied these effects by treating human AML cell lines with the LSD1 inhibitor bomedemstat and investigated the expression of antigen-presenting molecules by flow cytometry and quantitative PCR (qPCR). At a concentration of 100 nM, bomedemstat upregulated the expression of CIITA by a log2 fold change of 2.23 ± 0.68 (p < 0.01) in OCI-AML3, 2.17 ± 0.28 (p < 0.01) in HL60 and 1.80 ± 0.67 (p < 0.05) in MOLM-13. To assess if the observed upregulation of CIITA can lead to a re-expression of HLA-DR in these HLA-DR negative cell lines, we then treated these cell lines with 100 nM of bomedemstat with additional IFN-γ stimulation. Expression of HLA-DR and the co-stimulatory molecule CD86 was determined by flow cytometry. Bomedemstat increased the expression of HLA-DR and CD86 across all three cell lines leading to the generation of 26.63 ± 3.07% (p < 0.0001) HLA-DR +/CD86 + cells in OCI-AML3, 4.19 ± 1.68% (p < 0.001) HLA-DR +/CD86 + cells in HL-60 and 14.67 ± 2.03% (p < 0.001) HLA-DR +/CD86 + cells in MOLM-13. We further assessed the effects of bomedemstat treatment on cytokine production by qPCR. Notably, bomedemstat treatment increased the expression of CXCL-10 by a log2 fold change of 2.59 ± 0.66 (p < 0.001) in MOLM-13 and 4.12 ± 0.88 (p < 0.001) in HL-60 at 100 nM concentration. The upregulation of CXCL-10 was also validated using bead-based ELISA assays. To investigate the molecular mechanism of the observed concomitant upregulation of MHC-II molecules and CD86, we then used murine myeloid progenitor cells transformed by overexpression of Hoxa9 and Meis1 (H9M) or MN1. In H9M-transformed cells, bomedemstat concurrently upregulated MHC-II (I-A/I-E) and CD86 even in the absence of IFN-γ stimulation, leading to an increase in MHC-II +/CD86 + cells from 2.52 ± 1.22% at baseline to 29.95 ± 14.11 (p < 0.05). This effect was not observed in MN1-transformed cells. H9M-transformed progenitor cells from Irf8 knockout mice (H9M- Irf8-KO) did not show any upregulation of MHC-II, pointing towards a role for Irf8 in this process. To interrogate whether the concurrent upregulation of MHC-II and CD86 can enhance CD4 + T cell activation, we used ovalbumin-expressing H9M-transformed cells that were co-cultured with OT-II T cells after bomedemstat treatment. We could show that bomedemstat-treatment enhanced the ability of H9M-OVA cells to activate T cells as demonstrated by an enhanced expression of the T cell activation markers CD25 and CD69. Furthermore, bomedemstat also increased the immune killing of OVA-peptide pulsed H9M cells at an effector-to-target (E:T) ratio of 1:1 from 1.12 ± 4.01 to 19.70 ± 6.20% (p < 0.01). It also enhanced the killing of OVA-expressing H9M cells from 5.80 ± 2.95% to 17.70 ± 3.16% (p < 0.05). No significant killing effect was observed for MN1 cells. In conclusion, we demonstrate both phenotypically and functionally that LSD1 inhibition by bomedemstat can enhance the immunogenicity of human and murine AML models. These findings point towards a potential role of LSD1 inhibition as a maintenance therapy after allo-HSCT.

Sequential control of myeloid cell proliferation and differentiation by cytokine receptor-based chimeric antigen receptors.

As chimeric antigen receptor (CAR)-T cell therapy has been recently applied in clinics, controlling the fate of blood cells is increasingly important for curing blood disorders. In this study, we aim to construct proliferation-inducing and differentiation-inducing CARs (piCAR and diCAR) with two different antigen specificities and express them simultaneously on the cell surface. Since the two antigens are non-cross-reactive and exclusively activate piCAR or diCAR, sequential induction from cell proliferation to differentiation could be controlled by switching the antigens added in the culture medium. To demonstrate this notion, a murine myeloid progenitor cell line 32Dcl3, which proliferates in an IL-3-dependent manner and differentiates into granulocytes when cultured in the presence of G-CSF, is chosen as a model. To mimic the cell fate control of 32Dcl3 cells, IL-3R-based piCAR and G-CSFR-based diCAR are rationally designed and co-expressed in 32Dcl3 cells to evaluate the proliferation- and differentiation-inducing functions. Consequently, the sequential induction from proliferation to differentiation with switching the cytokine from IL-3 to G-CSF is successfully replaced by switching the antigen from one to another in the CARs-co-expressing cells. Thus, piCAR and diCAR may become a platform technology for sequentially controlling proliferation and differentiation of various cell types that need to be produced in cell and gene therapies.

Engraftment, Fate, and Function of HoxB8-Conditional Neutrophil Progenitors in the Unconditioned Murine Host.

The development and use of murine myeloid progenitor cell lines that are conditionally immortalized through expression of HoxB8 has provided a valuable tool for studies of neutrophil biology. Recent work has extended the utility of HoxB8-conditional progenitors to the in vivo setting via their transplantation into irradiated mice. Here, we describe the isolation of HoxB8-conditional progenitor cell lines that are unique in their ability to engraft in the naïve host in the absence of conditioning of the hematopoietic niche. Our results indicate that HoxB8-conditional progenitors engraft in a β1 integrin-dependent manner and transiently generate donor-derived mature neutrophils. Furthermore, we show that neutrophils derived in vivo from transplanted HoxB8-conditional progenitors are mobilized to the periphery and recruited to sites of inflammation in a manner that depends on the C-X-C chemokine receptor 2 and β2 integrins, the same mechanisms that have been described for recruitment of endogenous primary neutrophils. Together, our studies advance the understanding of HoxB8-conditional neutrophil progenitors and describe an innovative tool that, by virtue of its ability to engraft in the naïve host, will facilitate mechanistic in vivo experimentation on neutrophils.

CML-271: Neutrophil Extracellular Traps Are Increased in Chronic Myeloid Leukemia and Are Differentially Affected by Tyrosine Kinase Inhibitors

Context: Cardiovascular (CVS) complications are increasingly reported with the use of certain tyrosine kinase inhibitors (TKIs) to treat chronic myeloid leukemia (CML). Neutrophil extracellular traps (NETs) have previously been implicated in the pathogenesis of cancer-associated thrombosis. Objective: To assess NET formation in neutrophils from patients with CML, to characterize the role of reactive oxygen species (ROS) and peptidyl arginine deiminase 4 (PAD4) in NET formation in CML, and to assess the effects of TKIs on NET formation in CML. Design: Blood samples were obtained from patients with newly diagnosed CML and from age/gender-matched controls. NETs were assessed after stimulation with phorbol 12-myristate 13-acetate (PMA) or ionomycin (IO), with or without pre-treatment with imatinib, ponatinib, nilotinib, and dasatinib at clinically relevant concentrations. NET formation was assessed by morphology, immunofluorescence (IF), and the expression of proteins known to be associated with NET formation (NET-associated elastase, citrullinated histone H3 [H3cit], etc.). ER-HoxB8 conditionally immortalized murine myeloid progenitor cell line was differentiated into functional neutrophils and transduced with a BCR-ABL1 oncogene (or empty vector control). Results: Neutrophils isolated from treatment-naive patients with CML showed a significant increase in NET formation compared to matched controls at baseline and after stimulation with IO as assessed by IF and after stimulation with PMA as assessed by NET-associated elastase expression. Expression of H3cit, PAD4, and ROS was significantly higher in CML samples compared to controls. Pre-treatment of neutrophils with TKIs was associated with a differential effect on NET formation. Exposure to ponatinib significantly augmented NET-associated elastase and ROS levels as compared to control and other TKIs in neutrophils derived from patients with CML. BCR-ABL1 retrovirally transduced HoxB8-neutrophils demonstrated increased H3cit and myeloperoxidase expression, consistent with excess NET formation. This was inhibited by Cl-amidine, a PAD4 inhibitor, but not by the NADPH inhibitor diphenyleneiodonium (DPI). Ponatinib and nilotinib pre-exposure significantly increased H3cit expression in HoxB8-BCR-ABL1 cells after stimulation with IO. Conclusions: CML is associated with increased NET formation, which is further augmented by ponatinib and nilotinib, suggesting a possible role for NETs in promoting vascular toxicity in CML. In the ER-HoxB8-BCR-ABL1 model, NET formation is PAD4- but not ROS-dependent.

Tumor Suppressor, NORE1A (RASSF5), Is an Icsbp-Dependent Inhibitor of Aberrant Stress Granulopoiesis

Within the innate immune system, emergency (stress) granulopoiesis serves the specific and essential function of periodic granulocyte production in response to an infectious or inflammatory challenge. Emergency granulopoiesis occurs in four phases (immediate release of mature granulocytes from the bone marrow, expansion of the common granulocyte/monocyte progenitor pool, accelerated differentiation, and termination of the response) and requires IL1β, an IL1β-dependent increase in G-CSF and the transcription factors Stat3 and C/EPBβ. Our group has previously shown that interferon consensus sequence binding protein (Icsbp) is required for the termination of emergency granulopoiesis. Icsbp deficiency is associated with increased and sustained production of Fap1 and Gas2, aberrantly sustained granulocytosis and promotion of myeloid leukemia. We have identified a novel Icsbp target gene, NORE1A (RASSF5), that is likewise required for the termination of emergency granulopoiesis. Nore1A is a tumor suppressor that is down-regulated in many human cancers. We found that Icsbp acts as a NORE1A transcriptional activator and that Nore1A expression is decreased in bone marrow-derived myeloid progenitor cells from ICSBP -/- mice compared to wild-type (WT), and does not appropriately increase during ex vivo differentiation with G-CSF or IL1β. In cell culture, ICSBP -/- murine bone marrow cells were resistant to intrinsic or Fas-induced apoptosis compared to WT. However, transduction with a Nore1A expression vector rescued Fas-induced apoptosis in ICSBP -/- murine bone marrow-derived myeloid progenitor cells, associated with activation of the pro-apoptotic Mst1 kinase. We injected WT or NORE1 -/- mice intraperitoneally every 4 weeks with an ovalbumin/alum mixture (to stimulate emergency granulopoiesis) or saline control. NORE1 -/- mice demonstrated aberrantly exaggerated granulocytosis in response to repeated cycles of ovalbumin/alum stimulation compared to WT mice. NORE1 -/- mice also demonstrated a tendency to develop myeloid leukemia, even in the absence of overt immune stimulation. These findings suggest that Nore1A inhibits aberrant stress granulopoiesis by promoting apoptosis of myeloid cells in an Icsbp-dependent fashion. Our data implicate Nore1A deficiency in the deregulation of granulopoiesis and promotion of leukemogenesis that accompanies clinical scenarios associated with decreased Icsbp activity, such as human chronic myeloid leukemia. DisclosuresNo relevant conflicts of interest to declare.

Tolerance Induction by Myeloid Progenitor Cells Does Not Require Lethal Preconditioning or Hematopoietic Stem Cell Transplantation

Purpose Modulation of the immune system using cellular therapy is seen as an increasingly attractive option to prevent solid organ graft rejection, while maintaining sufficient immune function to prevent many of the complications associated with general immunosuppression. We have previously demonstrated that murine myeloid progenitor cells (mMPC), cryopreserved after expansion in vitro, induce robust tolerance for matched organ grafts when preconditioning included lethal whole body irradiation and third party hematopoietic stem cell transplantation (HSC). In this study we evaluated tolerance induced by mMPC when non-lethal pre-conditioning is used. Methods BALB/c or C57Bl/6 mice were pre-conditioned with various combinations of depleting antibodies, chemotherapeutic agents and immunosuppressants as well as infusion of 3 to 6 million mMPC expanded from HSC in vitro using a 10-day expansion protocol. mMPC were injected i.v. one or two weeks after skin graft placement. Skin grafts were either matched or not matched to the mMPC. These experiments looked at survival following the treatment without HSC transplantation, engraftment by mMPC-derived cells and tolerance to mMPC-matched, but not unmatched, skin grafts. Results The pre-conditioning combinations tested were non-lethal, and did not require HSC rescue (30-day survival without HSC: 44/45 mice). Tolerance to mMPC-matched grafts was varied with the protocol used and ranged from rejection of all grafts, rejection of most grafts to tolerance for 90% of the grafts. Using a protocol combining anti-thymocyte serum, anti-CD3 antibodies, busulfan, and rapamycin over a two week period allowed mMPC to be infused as late as two weeks after the skin transplants. 40 out of 44 grafts tested using this protocol survived for more than 5 months. Skin grafts that were not mMPC matched (mismatched or no mMPC) were all rejected (0/18). mMPC engraftment followed similar kinetics to those seen following lethal irradiation. Conclusion mMPC can prevent rejection of a mMPC-matched transplanted organ without the need for lethal whole body irradiation or HSC transplantation. This, combined with the ability to administer mMPC at least two weeks after the organ transplant (skin graft), opens up the possibility to develop clinical protocols that use fully matched organ donor-derived mMPC.

MiR-193a Is a Negative Regulator of Hematopoietic Stem Cells and Promotes Anti-Leukemic Effects in Acute Myeloid Leukemia

The microRNA (miRNA) family miR-193 consists of two members, miR-193a and miR-193b, which share identical seed regions and are therefore thought to be functionally redundant. However, the target spectrum of a miRNA is defined by the mRNA spectrum of the tissue, where it is expressed as well as dynamic strand preferences (miRNA-5p vs miRNA-3p) (Kuchenbauer et al., Blood, 2011). We and others previously showed that miR-193b is a tumor suppressor in acute myeloid leukemia (AML) (Bhayadia et al., JCO, 2018) and a potent regulator of hematopoietic stem cell expansion (Haetscher et al., Nat Com, 2015). However, the role of miR-193a in normal and malignant hematopoiesis is still unclear.First, we profiled the expression of miR-193a-5p and miR-193a-3p in human hematopoietic subpopulations from healthy donors via quantitative real-time PCR (qRT-PCR). Unlike miR-193b, which is enriched in hematopoietic stem cells (HSC) (Haetscher et al., Nat Com, 2015), miR-193a expression was restricted to differentiated cells of the myeloid lineage (granulocytes, monocytes, and erythrocytes). To investigate the function of miR-193a during normal hematopoiesis, we analyzed its effect on a murine myeloid progenitor cell line (32D cells) upon engineered overexpression of miR-193a in combination with granulocyte-stimulating growth factor (G-CSF) treatment (n=3-7). MiR-193a strongly promoted granulocytic differentiation of 32D cells already after two days compared to the control arm (p=0.006) as assessed by flow cytometry and morphological analysis. To analyze the effect of miR-193a on HSC function, we ectopically expressed miR-193a in highly purified E-SLAM (CD45+EPCR+CD48-CD150+) cells and transplanted them into lethally irradiated recipient mice (n=5 mice/arm). HSCs overexpressing miR-193a failed to reconstitute hematopoiesis (p=0.038). The negative effect on stem cell properties could be translated to human CD34+ cord blood (CB) cells, where miR-193a overexpression significantly reduced colony counts. Taken together, these results suggest, that miR-193a has pro-differentiation and anti-stemness functions.To assess the role of miR-193a in AML, we profiled miR-193a-5p and miR-193a-3p in two cohorts of de novo pediatric (n=187) and de novo adult AML patients (n=40) by qRT-PCR. We found that both miRNA strands (miR-193a-5p and miR-193a-3p) are present at similar levels, which is in contrast to the almost undetectable levels of miR-193b-5p strand, hinting at additional functional roles for miR-193a through its 5p arm. MiR-193a was significantly downregulated in adult and pediatric AML compared to healthy donor samples, suggesting a possible tumor suppressor function. To investigate the role of miR-193a in AML, we engineered transplantable, primary murine AML cell lines based on retroviral overexpression of Hoxa9/Meis1 (aggressive, short latency in vivo) with low endogenous miR-193a levels. Restoration of miR-193a by lentiviral overexpression delayed Hoxa9/Meis1 mediated leukemogenesis in vivo (p=0.01, n=8-10). Furthermore, miR-193a overexpression reduced leukemic growth of human AML cell lines (n=6) and decreased colony-forming capacity of primary AML patient samples (n=4; p=0.029) in vitro. To identify novel targets of miR-193a, we performed a proteomics screen in human NOMO1 AML cells overexpressing miR-193a compared to an empty control vector (n=5). We identified and verified stathmin (STMN1), a tubulin-associated, intracellular phosphoprotein previously linked to proliferation of AML cells, as a novel putative miR-193a target, further explaining its tumor suppressor effect.Taken together, we characterized miR-193a as a positive regulator of myeloid differentiation and negative modulator of HSCs. Based on the balanced presence of both miRNA arms, we hypothesize that each arm is functionally active and has different functions such as pro-differentiation and anti-stemness. Furthermore, this work is the first characterization of a miRNA family that exerts cooperative effects at both early and late hematopoietic differentiation stages, highlighting a novel mechanism of balancing anti-stemness and pro-differentiation. DisclosuresDöhner:Bristol Myers Squibb: Research Funding; AROG Pharmaceuticals: Research Funding; Sunesis: Consultancy, Honoraria, Research Funding; Seattle Genetics: Consultancy, Honoraria; Agios: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; Agios: Consultancy, Honoraria; Pfizer: Research Funding; Janssen: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; Astellas: Consultancy, Honoraria; Astex Pharmaceuticals: Consultancy, Honoraria; AROG Pharmaceuticals: Research Funding; Astellas: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Celator: Consultancy, Honoraria; Astex Pharmaceuticals: Consultancy, Honoraria; Bristol Myers Squibb: Research Funding; Celator: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Sunesis: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; Pfizer: Research Funding; Seattle Genetics: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding. Bullinger:Bristol-Myers Squibb: Speakers Bureau; Pfizer: Speakers Bureau; Bayer Oncology: Research Funding; Sanofi: Research Funding, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Honoraria, Speakers Bureau; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Speakers Bureau.

Paradoxical counteraction by imatinib against cell death in myeloid progenitor 32D cells expressing p210BCR-ABL.

Chronic myeloid leukemia (CML) is believed to be caused by the tyrosine kinase p210BCR-ABL, which exhibits growth-promoting and anti-apoptotic activities. However, mechanisms that allow cell differentiation in CML still remain elusive. Here we established tetracycline (Tet)-regulatable p210BCR-ABL-expressing murine 32D myeloid progenitor (32D/TetOff-p210) cells to explore p210BCR-ABL-induced cell death and differentiation. Tet-regulatable overexpression of p210BCR-ABL induced cell death due to the activation of both caspase-1 and caspase-3, coincident with the differentiation from myeloid progenitors into CD11b+Ly6C+Ly6G+ cells with segmented nuclei, exemplified as granulocytic myeloid-derived suppressor cells (G-MDSC), and the ability to secrete IL-1β, TNF-α, and S100A8/A9 into the culture supernatant. Treatment with imatinib almost completely abrogated all these phenotypes. Moreover, overexpression of a sensor of activated caspase-1 based on fluorescence resonance energy transfer (FRET) probe enabled us to detect activation of caspase-1 in a human CML cell line, K562. Furthermore, increased numbers of splenic G-MDSC associated with enhancement of S100A8/A9 production were observed in transgenic mice expressing p210BCR-ABL compared with that in wild-type mice. We also propose the novel mode of cell death in this 32D/TetOff-p210 system termed as myeloptosis.

The Impact of NFAT Inhibition on Neutrophil Effector Functions in Patients after Allogeneic Hematopoietic Stem Cell Transplantation and on Neutrophil Antifungal Defense and Myelopoiesis in Cyclosporin Α Treated and NFATc1LysM Mice

Background and Aims: Immunosuppressive medication e.g. by calcineurin inhibitors substantially contributes to the risk for opportunistic fungal infections in patients after allogeneic transplantation (HSCT). It is well known that the nuclear factor of activated T cells (NFAT) is an important transcription factor downstream of calcineurin especially in T cells. Additionally, recent data in rodent models indicate that NFAT also seems to play a relevant role in innate antifungal immune responses by polymorphonuclear neutrophils (PMN), as well as in regulation of myelopoiesis and myeloid differentiation.Methods: Firstly, isolated PMN from healthy donors were analyzed in vitro in absence or presence of CsA regarding their effector functions and activation-induced release of inflammatory mediators. Consecutively, blood samples of CsA-treated patients after allogeneic HSCT (n=17) and healthy donors (n=8) were analyzed ex vivo at two different time points as described above. Secondly, we used a murine IPA model (C57BL/6) and treated mice with CsA (18 mg/kg/d) or vehicle and challenged them with Aspergillus fumigatus (A. f.) conidia intratracheally. PMN recruitment to the lungs and pulmonary fungal clearance were examined by analyzing bronchoalveolar lavages (BAL) and peripheral blood (PB) using flow cytometry and cytometric bead array and murine lungs by fungal culture assays and histopathologic examination. Furthermore, survival was studied with neutropenic animals serving as positive controls. Moreover, LysM-specific NFATc1 knockout (NFATc1LysM) mice were bred lacking NFATc1 expression solely in myelomonocytic cells. These animals were also infected with A. f. and analyzed as further mentioned. In addition, we investigated myelopoiesis and myeloid differentiation by quantifying bone marrow derived myeloid progenitor cells from CsA treated or NFATc1LysMmice using flow cytometry and simultaneously counting PMN in PB under steady state conditions.Results: CsA enhanced phagocytosis of PMN in vitro and ex vivo in patients’ blood samples (54.2 % +/- 4.1 (patients) vs. 43.8 +/- 1.5, LPS, p=0.006). Moreover, PMNs migratory capabilities were reduced in vitro, whereas other effector functions or release of IL-8 were rather unaffected. PMNs of CsA-treated patients showed increased activation, degranulation and production of inflammatory mediators, but production of ROS was slightly decreased. In our in vivo model, IPA was lethal in neutropenic mice whereas solely CsA or vehicle treated mice survived the infection. CsA treatment resulted in enhanced PMN recruitment in BAL by trend, while pulmonary inflammation and PMN counts in PB remained stable. Indeed, fungal clearance was clearly constrained in CsA treated animals (2.1 x 105 CFU/lung +/- 0.5 (CsA) vs. 1.7 x 105 +/- 0.2, p<0.005). In our murine knockout model, NFATc1LysM mice infected with A. f. showed unimpaired survival without displaying detectable differences in PMN recruitment or fungal clearance. However, pulmonary inflammation and PMN counts in PB seemed to be more pronounced in knockout mice. Interestingly, BALs of CsA treated mice showed increased levels of IL-6 by trend (4634 pg/mL +/- 1073 (CsA) vs. 3108 +/- 729, p=0.48) but decreased levels of MCP-1 and TNF-α. In contrast, MCP-1, RANTES and TNF-α were enhanced by trend in BALs of NFATc1LysM mice, while IL-6 was reduced compared to wild type controls (3762 pg/mL +/- 729 vs. 4770 +/- 1613, p=0.81). PMN counts in PB were unaffected in NFATc1LysM mice but distribution of bone marrow derived murine myeloid progenitor cells was clearly impaired especially in megakaryocyte-erythroid progenitor cells (1.2 x 105 cells +/- 0.2 (NFATc1LysM) vs. 2.7 +/- 0.6, p=0.015), whereas solely CsA treatment had no influence.Conclusion: Results from our in vitro and ex vivo studies on patients' blood samples as well as from our murine in vivo IPA model indicate that NFAT regulates not only myelopoiesis, but also PMN functionalities in mice and humans. Nevertheless, these interactions are obviously multidimensional and potentially derive from involvement of different pathways. The underlying molecular mechanisms and clinical relevance of our findings in HSCT remain to be determined. DisclosuresNo relevant conflicts of interest to declare.

The Impact of NFAT Inhibition on Neutrophil Antifungal Defense and Myelopoiesis in Cyclosporine A Treated and NFATc1LysM Mice

Background and Aims: Immunodeficient patients after allogeneic stem cell transplantation (HSCT) are heavily threatened by opportunistic fungal infections like invasive pulmonary aspergillosis (IPA), partly due to immunosuppressive medication e.g. by calcineurin inhibitors like cyclosporine A (CsA) or tacrolimus. It is well known that the nuclear factor of activated T cells (NFAT) is an important transcription factor downstream of calcineurin in the adaptive immune system especially in T cells. Additionally, there is a growing body of evidence that NFAT also plays a substantial role in innate immune response against invasive fungal diseases by polymorphonuclear neutrophils (PMN), as well as in regulation of myelopoiesis and myeloid differentiation, as indicated by recent data in rodent models.Methods: Firstly, we used a murine IPA model (C57BL/6) to clarify the role of NFAT in antifungal innate immune response in vivo. To do so, we treated mice intraperitoneally with CsA (18mg/kg/d) or vehicle for 2 weeks and challenged them with Aspergillus fumigatus (A. f.) conidia intratracheally. 24 hours later, some mice were sacrificed and PMN recruitment to the lungs and pulmonary fungal clearance were examined by analyzing bronchoalveolar lavages (BAL) and peripheral blood (PB) by flow cytometry and murine lungs by fungal culture assays and histopathologic examination. In addition, survival of remaining infected mice was studied with neutropenic animals (by depletion with anti-Gr1) serving as positive controls. Secondly, LysM-specific NFATc1 knockout (NFATc1LysM) mice were bred lacking NFATc1 expression solely in myelomonocytic cells (i.e. PMN and monocytes). Furthermore, these animals were infected with A. f. and analyzed as described above. Secondly, we investigated myelopoiesis and myeloid differentiation under steady state conditions by quantifying bone marrow derived myeloid progenitor cells from CsA treated or NFATc1LysM mice using flow cytometry and simultaneously counting PMN in PB.Results: While the infection was lethal in CsA or vehicle treated neutropenic mice, all CsA or vehicle treated mice survived the infection. CsA treated mice showed enhanced PMN recruitment in BAL by trend (55.2% +/- 12.0 (CsA) vs. 33.7% +/- 8.0 (control), mean +/- SEM, p=0.053), whereas pulmonary inflammation and PMN counts in PB were comparable to controls. In contrast, fungal clearance was clearly impaired in animals after CsA treatment (2.1 x 105 CFU/lung after 48 hours +/- 0.5 (CsA) vs. 1.7 x 105 +/- 0.2 (control), p<0.005). Along with that, NFATc1LysM mice infected with A. f. showed unimpaired survival. However, there were no detectable differences in PMN recruitment or fungal clearance, whereas pulmonary inflammation and PMN counts in PB seemed to be more pronounced in knockout mice (1.0 inflammation points/lung +/- 0.12 (NFATc1LysM) vs. 0.7 +/- 0.07 (control), p=0.057; 1.5 x 103 PMN/µl +/- 0.2 (NFATc1LysM) vs. 0.9 +/- 0.1 (control), p=0.036). Distribution of bone marrow derived murine myeloid progenitor cells was unaffected through NFAT inhibition by CsA but clearly impaired in NFATc1LysM mice especially in megakaryocyte-erythroid progenitor cells (1.2 x 105 cells +/- 0.2 (NFATc1LysM) vs. 2.7 +/- 0.6, p=0.015) whereas PMN blood counts in PB were unchanged.Conclusions: In a mouse model, NFAT inhibition via treatment with CsA does not influence survival after infection with A. f. in vivo but affects PMN recruitment and local fungal clearance. To some extent this may be due to impaired PMN phagocytic and migratory capabilities as indicated by our in vitro and ex vivo studies (data not shown). However, solely NFATc1 downregulation in PMN apparently results in slightly different effects given that infected NFATc1LysM mice displayed enhanced pulmonary inflammation and elevated PMN blood counts compared to controls. Additionally, NFATc1 inhibition in NFATc1LysM mice leads to constrained myelopoiesis under steady state conditions without affecting peripheral PMN blood counts compared to untreated wild type controls. Further studies are needed to clarify underlying mechanisms and clinical relevance in HSCT of our findings. DisclosuresNo relevant conflicts of interest to declare.

Mtss1 is a critical epigenetically regulated tumor suppressor in CML.

Chronic myeloid leukemia (CML) is driven by malignant stem cells that can persist despite therapy. We have identified Metastasis suppressor 1 (Mtss1/MIM) to be downregulated in hematopoietic stem and progenitor cells from leukemic transgenic SCLtTA/Bcr-Abl mice and in patients with CML at diagnosis, and Mtss1 was restored when patients achieved complete remission. Forced expression of Mtss1 decreased clonogenic capacity and motility of murine myeloid progenitor cells and reduced tumor growth. Viral transduction of Mtss1 into lineage-depleted SCLtTA/Bcr-Abl bone marrow cells decreased leukemic cell burden in recipients, and leukemogenesis was reduced upon injection of Mtss1-overexpressing murine myeloid 32D cells. Tyrosine kinase inhibitor (TKI) therapy and reversion of Bcr-Abl expression increased Mtss1 expression but failed to restore it to control levels. CML patient samples revealed higher DNA methylation of specific Mtss1 promoter CpG sites that contain binding sites for Kaiso and Rest transcription factors. In summary, we identified a novel tumor suppressor in CML stem cells that is downregulated by both Bcr-Abl kinase-dependent and -independent mechanisms. Restored Mtss1 expression markedly inhibits primitive leukemic cell biology in vivo, providing a therapeutic rationale for the Bcr-Abl-Mtss1 axis to target TKI-resistant CML stem cells in patients.

Expression of JAK2-V617F Kinase Profoundly Dysregulates the Functional Properties of β1 Integrins on Myeloid Cells

Introduction: Deregulated signal transduction pathways driven by the constitutively active JAK2-V617F kinase, play a central role in pathogenesis of the classical Philadelphia negative myeloproliferative neoplasms (MPNs). Polycythemia vera, essential thrombocythemia and primary myelofibrosis are characterized by clonal proliferation of myeloid cells and development of inflammatory syndrome (CRP elevation, fever, high proinflammatory cytokine plasma levels) particularly, in advanced phases of the disease. Integrins are heterodimeric cell surface receptors that mediate a wide range of anchorage dependent events fundamentally essential in adhesion and mobility of leukocytes during inflammatory states. An intricate network of intracellular signaling pathways triggers the activation of intergins by allosteric conformational changes and/or by altering lateral mobility to increase receptor clustering.Methods: We investigated the role of JAK2-V617F in the regulation of β1 integrin activation, function and expression in myeloid cells using in vitro cell culture models, in vivo JAK2-V617F knock-in model, murine bone marrow transplantation model and finally, clinical patient samples (collected after informed consent obtained in accordance with the Declaration of Helsinki). Integrin activation was studied using flow cytomery based soluble ligand binding assay and by analyzing the expression of 9EG7 activation epitope. The functional static adhesion assay was performed on ligand coated plates and data was acquired using a fluorescence microplate reader. Surface expression of integrins was investigated by flow cytometry.Results: To identify a potential biological impact of JAK2-V617F on β1 integrins, we generated a 32D murine myeloid progenitor cell line transfected with EpoR and either wildtype JAK2 or JAK2-V617F. Under serum starved conditions, the presence of the oncogenic JAK2-V617F mutation strongly induced activation of β1 integrins as revealed by a 6-fold rise in binding to its soluble ligand, VCAM-1-Fc and enhanced exposure of the 9EG7 high-affinity conformation-specific epitope. This also translated into increased adhesion on VCAM-1 in comparison to wildtype cells. Importantly, inhibition of the constitutive JAK2-V617F kinase activity by JAK inhibitor treatment, significantly downregulated β1 integrin activation as well as cellular adhesion. Furthermore, shRNA based knockdown of the endogenous JAK2-V617F expression in HEL (Human Erythroleukemia) cells, reduced adhesion on VCAM-1, thereby underscoring the functional role of the JAK2-V617F kinase in the regulation of β1 integrins. Interestingly, the cell surface expression of β1 integrins remains unaltered in the presence of JAK2-V617F or inhibitor treatment in the 32D cells as well as, following genetic inhibition in HEL cells. Additionally, in an in vivo retroviral bone marrow transplantation model in Balb/c mice, the overexpression of wildtype or JAK2-V617F, showed similar β1 integrin expression in granulocytes. Currently, in vivo studies in a JAK2-V617F knock-in murine model are under way. We then extended our study to investigate the role of constitutively active JAK2-V617F in integrin function in MPN patients. Again, no significant difference was found in β1 integrin expression on primary granulocytes from JAK2-V617F positive MPN patients and age-matched healthy donors. However, granulocytes from JAK2-V617F-positive patients showed higher activation of β1 integrins as exhibited by a 6-fold increase in binding to soluble VCAM-1-Fc when compared to healthy donors. In addition, preliminary analysis suggests stronger adhesion of JAK2-V617F positive granulocytes on VCAM-1. In an attempt to dissect the underlying molecular mechanism, we uncovered a previously undescribed role of JAK2 in the inside-out signaling of integrin activation in 32D myeloid cells. Overexpression of wildype JAK2 or V617F resulted in activation of the small GTPase Rap1, a key integrin regulator, and was subsequently downregulated upon treatment with JAK inhibitor.Conclusions: Taken together, our study highlights a unique and important role of JAK2-V617F in regulation of β1 integrin activation in myeloid cells. This may have functional consequences in pathophysiology of the disease, particularly, in dysregulation of leukocyte adhesion during thrombosis and in induction of inflammation. DisclosuresNo relevant conflicts of interest to declare.

S‐Fms signalobody enhances myeloid cell growth and migration

Since receptor tyrosine kinases (RTKs) control various cell fates in many types of cells, mimicry of RTK functions is promising for artificial control of cell fates. We have previously developed single-chain Fv (scFv)/receptor chimeras named signalobodies that can mimic receptor signaling in response to a specific antigen. While the RTK-based signalobodies enabled us to control cell growth and migration, further extension of applicability in another cell type would underlie the impact of the RTK-based signalobodies. In this study, we applied the scFv-c-Fms (S-Fms) signalobody in a murine myeloid progenitor cell line, FDC-P1. S-Fms transduced a fluorescein-conjugated BSA (BSA-FL)-dependent growth signal and activated downstream signaling molecules including MEK, ERK, Akt, and STAT3, which are major constituents of Ras/MAPK, PI3K/Akt, and JAK/STAT signaling pathways. In addition, S-Fms transduced a migration signal as demonstrated by the transwell-based migration assay. Direct real-time observation of the cells further confirmed that FDC/S-Fms cells underwent directional cell migration toward a positive gradient of BSA-FL. These results demonstrated the utility of the S-Fms signalobody for controlling growth and migration of myeloid cells. Further extension of our approach includes economical large-scale production of practically relevant blood cells as well as artificial control of cell migration for tissue regeneration and immune response.

Increased Fanconi C expression contributes to the emergency granulopoiesis response

Emergency granulopoiesis is a component of the innate immune response that is induced in response to infectious or inflammatory challenge. It is characterized by the rapid expansion and differentiation of granulocyte/monocyte progenitor (GMP) populations, which is due in part to a shortened S-phase of the cell cycle. We found that IRF8 (also known as ICSBP), an interferon regulatory transcription factor that activates phagocyte effector genes during the innate immune response, activates the gene encoding Fanconi C (Fancc) in murine myeloid progenitor cells. Moreover, IRF8-induced Fancc transcription was augmented by treatment with IL-1β, an essential cytokine for emergency granulopoiesis. The Fanconi pathway participates in repair of stalled or collapsed replication forks during DNA replication, leading us to hypothesize that the Fanconi pathway contributes to genomic stability during emergency granulopoiesis. In support of this hypothesis, Fancc(-/-) mice developed anemia and neutropenia during repeated, failed episodes of emergency granulopoiesis. Failed emergency granulopoiesis in Fancc(-/-) mice was associated with excess apoptosis of HSCs and progenitor cells in the bone marrow and impaired HSC function. These studies have implications for understanding the pathogenesis of bone marrow failure in Fanconi anemia and suggest possible therapeutic approaches.

Hematopoietic Overexpression of the ETS Family Transcription Factor Erg, or the Oncogenic Fusion Protein TLS-ERG, Induces Erythro-Megakaryocytic Leukemia

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

Sensitivity of acute myeloid leukemia Kasumi-1 cells to binase toxic action depends on the expression of KIT and АML1-ETO oncogenes

Some RNases selectively attack malignant cells, triggering an apoptotic response, and therefore are considered as alternative chemotherapeutic drugs. Here we studied the effects of Bacillus intermedius RNase (binase) on murine myeloid progenitor cells FDC-P1; transduced FDC-P1 cells ectopically expressing mutated human KIT N822K oncogene and/or human AML1-ETO oncogene; and human leukemia Kasumi-1 cells expressing both of these oncogenes. Expression of both KIT and AML1-ETO oncogenes makes FDC-P1 cells sensitive to the toxic effects of binase. Kasumi-1 cells were the most responsive to the toxic actions of binase among the cell lines used in this work with an IC50 value of 0.56 µM. Either blocking the functional activity of the KIT protein with imatinib or knocking-down oncogene expression using lentiviral vectors producing shRNA against AML1-ETO or KIT eliminated the sensitivity of Kasumi-1 cells to binase toxic action and promoted their survival, even in the absence of KIT-dependent proliferation and antiapoptotic pathways. Here we provide evidence that the cooperative effect of the expression of mutated KIT and AML1-ETO oncogenes is crucial for selective toxic action of binase on malignant cells. These findings can facilitate clinical applications of binase providing a useful screen based on the presence of the corresponding target oncogenes in malignant cells.

Alcohol Suppresses the Granulopoietic Response to Pulmonary Streptococcus pneumoniae Infection with Enhancement of STAT3 Signaling

Enhanced granulopoietic activity is crucial for host defense against bacterial pneumonia. Alcohol impairs this response. The underlying mechanisms remain obscure. G-CSF produced by infected lung tissue plays a key role in stimulating bone marrow granulopoiesis. This study investigated the effects of alcohol on G-CSF signaling in the regulation of marrow myeloid progenitor cell proliferation in mice with Streptococcus pneumoniae pneumonia. Chronic alcohol consumption plus acute alcohol intoxication suppressed the increase in blood granulocyte counts following intrapulmonary challenge with S. pneumoniae. This suppression was associated with a significant decrease in bone marrow granulopoietic progenitor cell proliferation. Alcohol treatment significantly enhanced STAT3 phosphorylation in bone marrow cells of animals challenged with S. pneumoniae. In vitro experiments showed that G-CSF-induced activation of STAT3-p27(Kip1) pathway in murine myeloid progenitor cell line 32D-G-CSFR cells was markedly enhanced by alcohol exposure. Alcohol dose dependently inhibited G-CSF-stimulated 32D-G-CSFR cell proliferation. This impairment of myeloid progenitor cell proliferation was not attenuated by inhibition of alcohol metabolism through either the alcohol dehydrogenase pathway or the cytochrome P450 system. These data suggest that alcohol enhances G-CSF-associated STAT3-p27(Kip1) signaling, which impairs granulopoietic progenitor cell proliferation by inducing cell cycling arrest and facilitating their terminal differentiation during the granulopoietic response to pulmonary infection.

Enforced Expression of Mir-125b Blocks Granulocytic Differentiation by Simultaneous Repression of Multiple Targets.

AbstractAbstract 1554Granulopoiesis is a multistep process controlled by a complex system of cytokines and transcription factors that modulate expression of downstream genes and mediate proliferation and differentiation signals. Recent findings demonstrate that miRNAs may provide an additional level of control. We used the 32D murine myeloid progenitor cell line as a model system to study G-CSF-induced granulocytic differentiation. Based on miRNA-expression analyzed by microarray and miR-qRT-PCR, we identified several miRNAs (including miR-34a-c, -125b, -155, 181b, 223, 291a, 370) potentially involved in regulation of granulocytic differentiation. To define the role of individual miRNAs, stable gain- and loss-of-function phenotypes were generated using lentiviral gene transfer of pre-miRNAs and antagomiRs, respectively. We found that enforced expression of miR-125b in undifferentiated 32D myeloid precursors resulted in a complete block of granulocytic differentiation upon G-CSF treatment and partially protected the cells from IL-3 withdrawal-induced cell death. The pivotal role of miR-125b in myeloid differentiation was demonstrated in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) with the chromosomal translocation t(2;11)(p21;q23) resulting in miR-125b over-expression [Bousquet M, et al. JEM 2008]. Furthermore, clonogenic assays of primary Lin- cells revealed that miR-125b cultures generated more and larger colonies as compared to miR-control, indicating a proliferative advantage of miR-125b over-expressing progenitor cells. Correspondingly, enforced miR-125b expression in murine bone marrow has recently been shown to induce a lethal myeloproliferative disorder (MPD) in transplanted mice [O'Connell RM, et al. PNAS 2010]. However, the molecular mechanisms mediated by miR-125b in hematopoietic cells remain largely unknown. By utilizing different miRNA-target prediction programs including Target Scan, DIANA microT v 0.3 and RNA22, we identified Bcl-2 family members Bak1, Mcl-1, Bmf and Puma as putative targets of miR-125b. We confirmed diminished Bak1 protein expression in 32D cells (reduction by ∼40%); however, lentivirus-mediated RNAi targeting Bak1 to the similar level as induced by enforced miR-125b expression resulted only in a delay of cell death in the presence of G-CSF but not in granulocytic differentiation block. Further computational analysis revealed two putative miR-125b binding sites in 3' UTR of Stat3, the principal Stat protein activated following G-CSF treatment and robustly involved in myeloid differentiation. Specific binding of miR-125b to Stat3 3'UTR was validated by luciferase reporter assay and confirmed by western blotting (reduction by ∼30-40%). We found that shRNA-mediated strong reduction of Stat3 protein (reduction by ∼80%) enables G-CSF dependent cell proliferation (with a delay of some days), and induces a complete block of cellular differentiation. On the contrary, a mild reduction of Stat3 protein expression(by ∼30–40%), as induced by enforced expression of miR-125b, resulted only in a slight delay of cell death but not in block of granulocytic differentiation. Interestingly, simultaneous reduction of both Bak1 and Stat3 by combinatorial RNAi to similar levels as observed in the presence of enforced miR-125b expression, cooperatively delays cell death upon G-CSF treatment as compared to knock-down of Bak1 or Stat3 solely. Since miR-125b has approximately 500 predicted target genes and neither mild knock-down of Bak1 nor Stat3 alone resulted in a block of granulocytic differentiation, we may either miss an additional/unknown important target or the miR-125b-induced phenotype requires simultaneous repression of more targets in addition to Stat3 and Bak1. Nevertheless our study provides experimental evidence that miR-125-mediated phenotypes arise from mild and simultaneous down-regulation of multiple targets/signalling pathways. Therefore the precise and quantitative analysis of miRNA-targets is required to evaluate the safety and benefit of eventual miRNA-based therapeutic strategies to modulate complex cellular phenotypes. Disclosures:No relevant conflicts of interest to declare.

Phosphorylation of MLL by ATR is required for execution of mammalian S-phase checkpoint

Cell cycle checkpoints are implemented to safeguard the genome, avoiding the accumulation of genetic errors. Checkpoint loss results in genomic instability and contributes to the evolution of cancer. Among G1-, S-, G2- and M-phase checkpoints, genetic studies indicate the role of an intact S-phase checkpoint in maintaining genome integrity. Although the basic framework of the S-phase checkpoint in multicellular organisms has been outlined, the mechanistic details remain to be elucidated. Human chromosome-11 band-q23 translocations disrupting the MLL gene lead to poor prognostic leukaemias. Here we assign MLL as a novel effector in the mammalian S-phase checkpoint network and identify checkpoint dysfunction as an underlying mechanism of MLL leukaemias. MLL is phosphorylated at serine 516 by ATR in response to genotoxic stress in the S phase, which disrupts its interaction with, and hence its degradation by, the SCF(Skp2) E3 ligase, leading to its accumulation. Stabilized MLL protein accumulates on chromatin, methylates histone H3 lysine 4 at late replication origins and inhibits the loading of CDC45 to delay DNA replication. Cells deficient in MLL showed radioresistant DNA synthesis and chromatid-type genomic abnormalities, indicative of S-phase checkpoint dysfunction. Reconstitution of Mll(-/-) (Mll also known as Mll1) mouse embryonic fibroblasts with wild-type but not S516A or ΔSET mutant MLL rescues the S-phase checkpoint defects. Moreover, murine myeloid progenitor cells carrying an Mll-CBP knock-in allele that mimics human t(11;16) leukaemia show a severe radioresistant DNA synthesis phenotype. MLL fusions function as dominant negative mutants that abrogate the ATR-mediated phosphorylation/stabilization of wild-type MLL on damage to DNA, and thus compromise the S-phase checkpoint. Together, our results identify MLL as a key constituent of the mammalian DNA damage response pathway and show that deregulation of the S-phase checkpoint incurred by MLL translocations probably contributes to the pathogenesis of human MLL leukaemias.