WT Bone Marrow Research Articles

The role of thromboxane prostanoid receptor signaling in gastric ulcer healing.

The process of gastric ulcer healing includes cell migration, proliferation, angiogenesis and re‐epithelialization. Platelets contain angiogenesis stimulating factors that induce angiogenesis. Thromboxane A2 (TXA2) not only induces platelet activity but also angiogenesis. This study investigated the role of TXA2 in gastric ulcer healing using TXA2 receptor knockout (TPKO) mice. Gastric ulcer healing was suppressed by treatment with the TXA2 synthase inhibitor OKY‐046 and the TXA2 receptor antagonist S‐1452 compared with vehicle‐treated mice. TPKO showed delayed gastric ulcer healing compared with wild‐type mice (WT). The number of microvessels and CD31 expression were lower in TPKO than in WT mice, and TPKO suppressed the expression of transforming growth factor beta (TGF‐β) and vascular endothelial growth factor A (VEGF‐A) in areas around gastric ulcers. Immunofluorescence assays showed that TGF‐β and VEGF‐A co‐localized with platelets. Gastric ulcer healing was significantly reduced in WT mice transplanted with TPKO compared with WT bone marrow. These results suggested that TP signalling on platelets facilitates gastric ulcer healing through TGF‐β and VEGF‐A.

Abstract MP17: TLR4 May Play A Role In Accelerated Atherosclerosis In Beta Thalassemia

Beta Thalassemia (BT) is an autosomal recessive hemoglobinopathy that affects 80 million people worldwide. Children with BT display an increase in carotid-intima media thickness, an early sign suggestive of premature atherosclerosis. However, it is unknown if there is a direct relationship between BT and atherosclerotic disease. BT has increased free heme leading to ROS generation directly or via Toll Like Receptor (TLR4) activation, but the role of heme in disease progression in BT is poorly characterized. The aim of this study is to evaluate the progression of atherosclerosis in BT mice and the potential role of TLR4. Atherosclerosis was induced by implementing short (ST) and long term (LT) models. In our ST model, both BT ( Hbb th-3 ) and wild type (b6) male/female littermates (n=5) were fed a Paigen high fat diet (PD) for 8 weeks. On week 2, mice were injected with AAV8-PCSK9 to knockdown the LDL receptor. On weeks 4-8, mice were treated with Angiotensin ll via subcutaneous osmotic pumps implantation. The LT model was performed by placing mice on 3 months of PD with PCSK9 overexpression. Atherosclerosis was evaluated by atherosclerotic plaque lesion area of the descending aorta via en face analysis, histology of the aortic roots, and RT-qPCR of inflammatory markers. In addition, to evaluate the possible contribution of TLR4 in atherosclerosis, bone marrow transplants were performed by grafting WT and BT bone marrow (n=2-4) to both b6 and TLR4 -/- mice, and atherosclerosis was performed as described in our LT model.In our ST model, aortic en face analysis revealed elevated plaque accumulation in BT mice (WT: 22 ± 4% plaque area vs. Thal: 45 ± 11% plaque area, p = .024). Aortic root (H&E) analysis did not display a significant difference in our ST model, but it was significantly increased in the LT model. Gene expression profiling indicated a significant increase in both IL-6 and CD-68 (p<0.041) in BT mice. In our BMT experiments, we saw a significant decrease in aortic root plaque in TLR4 -/- mice with Thal BM (.24 ± .06 mm 2 ) compared to wild type mice with Thal BM (.39 ± .07 mm 2 ). Our data demonstrate for the first time that the underlying pathophysiology of BT clearly leads to accelerated atherosclerosis and suggest TLR4 is playing a role in atherosclerotic development in BT.

Abstract 55: Hiv Viral Proteins Elevate Blood Pressure And Impairs Endothelial Function In Resistance Arteries Via Sex Specific, Immune And Nox1-dependent Mechanisms In Mice.

Thanks to the onset of combination antiretroviral therapy (cART), patients living with HIV live longer but experience accelerated rates of hypertension. However, the etiology of HIV-associated hypertension remains ill defined, specifically the respective contributions of repressed viral infection and cART treatment. Herein, we took advantage of a transgenic model (Tg26) of repressed viral infection to investigate the contribution of HIV viral proteins to hypertension. Quantification of inflammatory cytokines revealed elevated circulating TNFα levels but also high aorta and intraperitoneal immune cells TNFα in male Tg26 mice leading to the hypothesis that HIV-associated hypertension involves immune-derived TNFα secretion and endothelial dysfunction in males and females Tg26 mice. Blood pressure (BP) was measured via radio telemetry and vascular reactivity studies performed via wire myography. BP analysis revealed increased mean arterial pressure (MAP: male: WT=112.3±1.3 vs Tg26=121.9±4.0 mmHg/ female: WT=110.6±3.01/ Tg26=120.3±6.9 mmHg) and heart rate in both sexes (P<0.05). However, blockade of TNFα action with etanercept restored BP and aortic endothelial function in male Tg26 mice only. Along with an increase in TNFα, male Tg26 aortas showed infiltration of Tcells and proinflammatory cytokines IL-18, and IL-6, which were not seen in females. However, scavenging of reactive oxygen species with the selective NOX1 inhibitor GKT771 restored aorta endothelial function in both sexes. A contributor to hypertension is impaired endothelial relaxation in resistance arteries. Mesenteric arteries showed impaired endothelial relaxation to acetylcholine in both male and female Tg26 mice (P<0.05) with no impairment in smooth muscle cell relaxation. This phenotype was reproduced in WT mice transplanted with Tg26 bone marrow (BM) and remarkably reversed in Tg26 mice receiving WT BM supporting a clear role for immune cells in endothelial dysfunction. Collectively, these data indicate that HIV-related hypertension involves immune and NOX1-dependent endothelial dysfunction in both sexes but TNFα-dependent mechanisms in males only providing evidence of sex specific mechanisms.

Tet2 Controls the Responses of \u03b2 cells to Inflammation in Autoimmune Diabetes

β cells may participate and contribute to their own demise during Type 1 diabetes (T1D). Here we report a role of their expression of Tet2 in regulating immune killing. Tet2 is induced in murine and human β cells with inflammation but its expression is reduced in surviving β cells. Tet2-KO mice that receive WT bone marrow transplants develop insulitis but not diabetes and islet infiltrates do not eliminate β cells even though immune cells from the mice can transfer diabetes to NOD/scid recipients. Tet2-KO recipients are protected from transfer of disease by diabetogenic immune cells.Tet2-KO β cells show reduced expression of IFNγ-induced inflammatory genes that are needed to activate diabetogenic T cells. Here we show that Tet2 regulates pathologic interactions between β cells and immune cells and controls damaging inflammatory pathways. Our data suggests that eliminating TET2 in β cells may reduce activating pathologic immune cells and killing of β cells.

The Macrophage Nuclear Receptor Coactivator 4 Counteracts Iron-Deficiency Anemia

The Nuclear Receptor Coactivator 4 (NCOA4) protein has been recently recognized as a novel player in iron metabolism contributing to regulation of cell and systemic iron homeostasis. NCOA4 is a cargo receptor that promotes selectively autophagy of the iron storage ferritin in conditions of iron deficiency, facilitating iron recovery from cellular stores. Inactivation of NCOA4 in cells increases ferritin aggregates [Mancias JD et al., Nature 2014; Dowdle WE et al., Nat Cell Biol 2014; Mancias JD et al., Elife 2015] and C57Bl/6 Ncoa4-ko mice show ferritin and iron accumulation in several organs, in particular spleen [Dowdle WE et al., Nat Cell Biol 2014; Bellelli R et al., Cell Rep 2016]. In these animals, iron retention in tissue ferritin reduces iron availability for erythropoiesis causing mild microcytic anaemia in iron-balanced (IB) conditions. In addition, compared to wt animals, Ncoa4-ko mice fed an iron-deficient (ID) diet develop a more severe anemia [Bellelli R et al., Cell Rep 2016].Given the important impact of mouse strain on iron metabolism, we evaluated the ability of Ncoa4-ko mice on a different background to respond to variation of dietary iron content. To this aim wt and null mice on Sv129/J background were fed an IB or an ID diet for 6 months. The ID diet induced almost no changes in hemoglobin levels of wt mice (IB=14.0±1.9 g/dL; ID=13.3±2.9 g/dL), while Ncoa4-ko developed severe microcytic anemia (Hb: IB=13.5±2.0 g/dL, ID=6.7±2.9 g/dL, p<0.05; MCV: IB=47.8±1.3 fL, ID=36.5±4.8 fL, p<0.05). These results are consistent with wt mice on Sv129/J strain being able to counteract ID by using the large reserves of iron present in their tissues. Notwithstanding such high iron deposits, Sv129/J Ncoa4 -ko fail this compensation. To better evaluate the bone marrow (BM)-derived cells (erythroid precursors, BM and spleen macrophages) contribution to the inability of Ncoa4-ko mice to respond to variation of dietary iron in comparison to other tissues (eg. liver and duodenum), we performed BM transplantation experiments in Ncoa4-ko using Sv129/J background. Lethally irradiated Ncoa4-ko mice were transplanted with wt or Ncoa4-ko BM, generating chimeric null animals with wt (Ncoa4-kowt-BM) or Ncoa4-ko BM cellsas control (Ncoa4-koko-BM). Previously published results obtained in vitro and in vivo in zebrafish suggest an essential role for NCOA4 in erythroid cells development and hemoglobinization [Mancias JD et al., Elife 2015; Ryu MS et al., JCI 2017]. However, we observed that Ncoa4-ko BM cells were able to reconstitute an almost normal erythropoiesis in lethally irradiated mice, indicating that in vivo the loss of Ncoa4 has a limited, if any, effect on the erythroid lineage differentiation. In addition, Ncoa4-ko mice with wt bone marrow have normal hematological parameters, suggesting that the mild microcytosis observed in Ncoa4-ko animals in basal conditions is due to a defect of BM-derived cells. Theoretically this effect may be due to cells belonging either to the erythroid or to the macrophage lineage; based on our findings we suggest a prevalent role for macrophages. In line with this hypothesis, Ncoa4-koko-BM mice fed an ID diet for 3 months developed a more severe anemia than Ncoa4-kowt-BM (Hb= 10.7±0.6 g/dL vs 11.7±1.0 g/dL; p=0.06), accompanied by comparable liver (LIC: Ncoa4-koko-BM=174.8±50.4 μg/g; Ncoa4-kowt-BM=157.1±49.5 μg/g) but higher spleen iron concentration (SIC: Ncoa4-koko-BM=719.0±221.3 μg/g; Ncoa4-kowt-BM=176.1±74.6 μg/g; p<0.05), consistent with decreased iron recycling by spleen macrophages of Ncoa4-koko-BM animals.Over all, our results suggest that NCOA4-mediated ferritinophagy plays a central role in macrophages in counteracting dietary ID, promoting iron release and ensuring efficient erythropoiesis. DisclosuresCamaschella:Vifor Pharma: Membership on an entity's Board of Directors or advisory committees.

Wave-Complex-Dependent Actin Dynamics Controls Hematopoietic Stem Cell Bone Marrow Retention and Orderly Differentiation into Mature Hematopoietic Lineages

BackgroundSpatiotemporally controlled actin dynamics are crucial for a vast number of cellular functions including migration, proliferation, attachment or immunological synapse formation. Environmental signals activate cognate surface receptors which in turn engage specific small GTPases. Signal integrators (e.g. WASP, WAVE complex or WIP) propagate the signal to Arp2/3 complex and lead to immediate actin polymerization. Here we show that disruption of one of the actin polymerization pathways through specific targeting of WAVE complex disturbs hematopoietic stem and progenitor cells (HSPC) bone marrow retention and their sequential differentiation towards mature blood cells.MethodsWe made use of the recently described mouse model of Hem1 ablation. Hem1 is a strictly hematopoietic specific component of WAVE complex; its loss disrupts the entire WAVE complex exclusively in cells of hematopoietic origin. An array of in vitro assays was employed to test cell intrinsic effects of Hem1 ablation on actin polymerization dependent cellular functions. In vivo transplantation experiments were performed to study the impact of Hem1 ablation on immature and in consequence on mature hematopoiesis.ResultsHem1-/- HSPC display a bone marrow (BM) retention defect evidenced by strongly elevated numbers of circulating HSPC and 50% reduced BM cellularity. In line with that, migration towards CXCL12, a key BM anchor for HSPC, was completely abrogated in Hem1-/- HSPC. Attachment to feeder cells was also decreased by 35% compared to WT HSPC, although stress fiber formation upon CXCL12 stimulation or general ability to polarize was not affected. Next we tested whether this cellular phenotype had any impact on HSPC performance in vivo. Homing, the initial interaction of transplanted HSPC with BM environment, was not affected by Hem1 ablation, while engraftment, the ability to repopulate all mature hematopoietic lineages, was strongly diminished. Platelet and RBC engraftment was delayed, whereas Hem1-/- neutrophils did not engraft at all (Figure 1). Hem1-/- mice presented with chronic inflammation, hyperleukocytosis and elevated pro-inflammatory cytokine plasma levels. To rule out inflammation as causal for numeric and functional Hem1-/- HSPC depletion we performed co-transplantation experiments. Hem1-/- or WT BM was transplanted in a four-fold excess over GFP+ competitor BM. In Hem1-/- transplanted recipients, already 4 weeks after transplantation 60 to 90% of both myeloid and lymphoid compartments were GFP+, whereas, as expected, in the control experiment WT BM out-competed GFP+ BM over time. A similar picture emerged in secondary recipients. Again Hem1-/- BM completely lost against, whereas WT BM prevailed over GFP+ competitor. We furthermore confirmed that the Hem1-/- HSPC cell intrinsic defect is already apparent during embryonic development: As in BM of adult Hem1-/- mice, HSPC content in E14.5 old fetal livers (FL) was reduced by half compared to WT littermates. Also the competitiveness of FL HSPC was strongly impaired, similar to Hem1-/- adult BM HSPC. Whereas short term engraftment was provided by more mature competitor GFP+ BM in both recipients groups, already 4 weeks after transplantation, as expected, WT FL derived HSPC took over blood cell production and completely outcompeted GFP+ BM. In Hem1-/- FL recipients, by contrast, the contribution of competitor GFP+ BM to hematopoiesis remained high for the whole period tested without significant contribution of Hem1-/- FL hematopoiesis (Figure 2). Taken together, Hem1 (and, by extension, WAVE complex) governs HSPC BM retention and proper engraftment. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Modulating Hedgehog Signaling Reduces MAPK, TGF-β, and Marrow Fibrosis in a Transgenic Mouse Model of JAK2V617F Associated Myeloproliferative Disease

Treatment of JAK2V617F driven myeloproliferative neoplasms (MPN) with FDA approved drug Ruxolitinib (JAK inhibitor, JAKi) has shown limited benefits. The failure of JAKi to eradicate JAK2V617F disease has led to the search for other pathways as potential therapeutic targets for this disease. Hedgehog (Hh) pathway inhibitors targeting Smoothened (SMO) have shown promising activity in MPN by reducing bone marrow fibrosis but are inadequate as single agents.In the present study we tested the effects of SMO inhibitor, PF-04449913 in transgenic mouse model of myeloproliferative disease (vavJak2V617F, gifted by Joe Zhao, University of Oklahoma Health Sciences Center). JAK2V617F transgenic mice exhibit a number of phenotypes that recapitulate those observed in MPN including bone marrow fibrosis and splenomegaly.Treatment with SMO inhibitor, PF-04449913, reduces splenomegaly. JAK2V617F mice showed pronounced splenomegaly compared to WT mice and treatment with SMO inhibitor resulted in a significant decrease in spleen size (Figure 1A). This was also associated with a drop in IL-4 and IL-7 in the peripheral blood (data not shown).SMO inhibitor reduces JAK2V617F allelic burden. In the next set of experiment we made mixed chimeras by transplanting one million JAK2V617F or WT bone marrow into lethally irradiated normal CD45.1 congenic hosts. Transplanted recipient mice were treated with PF-04449913 for one month and assessed for changes in peripheral blood chimerism. Treatment with PF-04449913 resulted in no change in WT engraftment. However, we found that PF-04449913 reduces the JAK2V617F allele burden in 3/5 mice.JAKV617F activates Hh signaling. Proteins were isolated from the spleens of 8-10 week JAK2V617F or WT mice. Western blot analyses demonstrate that JAK2V617F splenocytes express high SHH levels. We next measured levels of the GLI activator (GLI1) and the GLI repressor (GLI3R). Activation of Hh signaling is associated with an increase in GLI1 and a decrease in GLI3R. Treatment of the mice with PF-04449913 by daily gavage at 100mg/kg reduced GLI1 and increased GLI3R.JAK2V617F is associated with increased pERK, AKT, NF-κB activation. Oh and colleagues recently remonstrated the MPN were associated with activation of MAPK and NF-κB signaling pathways (Fisher et al., Leukemia, 2017). To understand how Hh signaling interacted with these pathways, we assessed ERK, AKT, and NF-κB activity by western blotting. JAK2V617F mice showed increased pERK, AKT and NFKB signaling. Treatment with PF-04449913 reduced pERK and AKT levels, confirming that both ERK and AKT are downstream of SMO, while it had little effect on IκBα levels suggesting that NF-κB activation occurs upstream of SMO.SMO inhibitor blocks bone marrow fibrosis by reducing TGF-β signaling. Numerous studies have implicated TGF-β in the pathogenesis of fibrosis. We analyzed the bone marrow of patients with JAK2V617F MPN and found them to express high TGF-β levels (Figure 1B). These findings were recapitulated in the JAK2V617F transgenic mice with levels of TGF-β in JAK2V617F splenocytes 15-fold greater than WT mice. Treatment with PF-04449913 normalized the levels of TGF-β (Figure 1C). Using a OP-9 co-culture system we demonstrated that JAK2V617F splenocytes robustly induced pSMAD2, which was completely blocked by PF-04449913 (Figure 1D). To assess for changes in bone marrow fibrosis, mice at 27 weeks were started on daily treatment with PF-04449913 for one month. Treatment with PF-04449913 blocks bone marrow fibrosis as demonstrated by decreased reticulin staining and reduced hydroxyproline. (Figure 1E-F).In conclusion, these data are the first to report that the Smoothened inhibitor, PF-04449913 modulates TGF-β and MAPK signaling in MPN and this results in decreased splenomegaly and marrow fibrosis. These results provide critical insight into the mechanism of action of SMO inhibitors in JAK2V617F associated MPN, and support the rational development of SMO and JAK inhibitor combinations in MPN.Figure Legend(A) SMO antagonist, PF-04449913 reduces splenomegaly. (B) Bone marrow of JAK2V617F MPN patient express high TGF-β levels. (C) Splenocytes of JAK2V617F transgenic mice express high TGF-β levels and PF-04449913 treatment normalizes TGF-β levels. (D) JAK2V617F induces Hh dependent TGF-β/pSMAD2 signaling in stroma. (E-F) PF-04449913 blocks fibrosis as measured by reticulin staining and quantification of Hydroxyproline. [Display omitted] DisclosuresO'Connell:Astex Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees; Otsuka: Membership on an entity's Board of Directors or advisory committees. Merchant:Pfizer: Consultancy, Research Funding.

Bcr-Abl1 Kinase-Independent Upregulation of FcγRIIb Mediates Malignant Signaling That Is Critical for Leukemogenesis

Chronic myeloid leukemia (CML) is provoked by the chromosomal translocation t(9;22) within the hematopoietic stem cell (HSC) compartment, and this mutation gives rise to the oncogenic tyrosine kinase Bcr-Abl1. Although tyrosine kinase inhibitor (TKI) therapy results in enormous clinical success, TKIs fail to eradicate the disease initiating leukemic stem cell population (LSC) in the majority of CML patients. In a transgenic CML mice (SCLtTA/Bcr-Abl), which we have previously demonstrated to serve as an excellent model to study Bcr-Abl1 independent stem cell persistence, we identified the ITIM carrying Fc gamma receptor IIb (FcγRIIb; CD32) to be 2.8-fold upregulated in Bcr-Abl1+ versus control bone-marrow (BM) derived LSK (lin-;Sca-1+;c-kit+) cells, using microarray and qRT-PCR. Aiming to evaluate the role of FcγRIIb function in Bcr-Abl1 mediated leukemogenesis, we previously retrovirally infected 5-FU treated SCLtTA/Bcr-Abl BM cells (CD45.1+) using FcγRIIb shRNA or scrambled control and transplanted these cells into FVB/N wildtype (WT) CD45.2+ recipients. Spleen weight in recipients receiving shRNA transduced stem and progenitor cells was significantly reduced (352 ± 59.1 mg), as compared to scrambled controls (568.1 ± 101.7 mg). Stem cell compartment analysis revealed decreased leukemic cells of shRNA versus scrambled control transplanted mice in the BM LSK compartment (lin-, c-kit+, Sca-1+, CD45.1+, GFP+, 1.38-fold, p≤0.05). In addition, these effects were even more pronounced upon total depletion of FcγRIIb using the retroviral Bcr-Abl1 model. In these mice, complete loss of FcγRIIb decreased malignant BM LSK cells by 3.5-fold (p<0.05) as compared to Bcr-Abl1 transduced FcγRIIb+/+recipients. Here, we have now studied the oncogenic mechanisms of FcγRIIb expression using the transgenic mice, a retroviral CML mouse model, as well as human cells.Ectopic Bcr-Abl1 expression in human TF-1 cells increased FcγRIIb levels by 4-fold (p<0.001). In lin- SCLtTA/Bcr-Abl BM cells, FcγRIIb was increased 1.4-fold (p<0.001), and receptor upregulation was thus higher in the LSK compartment (2.8-fold) as compared to lin- cells. We then studied the effect of TKI treatment on FcγRIIb expression in Bcr-Abl1 transduced lin- BM and lin-SCLtTA/Bcr-Abl cells using imatinib or dasatinib. However, both TKIs did not reduce FcγRIIb levels, suggesting that persistent receptor upregulation is regulated independently of the Bcr-Abl1 kinase. Next, we used HoxB8-immortalized progenitor cells from FcγRIIb-/- vs wt BM and virally introduced Bcr-Abl1 or ev. CFU (colony forming unit) potential in Bcr-Abl1 positive FcγRIIb -/- cells was significantly decreased 4-fold (p<0.01) as compared to Bcr-Abl1 transduced wt cells. Interestingly, re-introduction of FcγRIIb into Bcr-Abl1 positive FcγRIIb-/- cells significantly increased CFU capacity again. CFU reduction was also observed upon overexpression of an SH2-SHIP1 domain in leukemic cells, as SHIP1 is the predominant FcγRIIb binding partner upon activation. Furthermore, depletion of the receptor, using shRNAs, decreased the proliferation rate in CML cells (2.3-fold on day 4, p<0.01) and this was accompanied by an increase in G0/G1 phase and decrease in S and G2/M phase in CML cells lacking the receptor. Along the same line, expression of p16, p19 and p21 were significantly increased in FcγRIIb depleted CML cells. CFU potential as well as proliferation was likewise decreased (10-fold, p<0.01; 2.8-fold p<0.01) in shRNA-mediated FcγRIIb knock-down immortalized SCLtTA/Bcr-Abl CML cells. Western-Blot analysis showed that FcγRIIb is phosphorylated in leukemic cells, and analysis of downstream signal pathways revealed decreased levels of p-ERK and p-p38 among others in FcγRIIb-/-, compared to FcγRIIb+/+ Bcr-Abl1 transduced cells. Taken together, we demonstrate that the FcγRIIb upregulation on LSC from transgenic CML mice, progenitor cells and human cells critical for CML cell biology. Genetic depletion or knock-down of the receptor reduces CFU capacity and cell growth which is mediated due to impaired leukemic downstream signaling. As FcγRIIb inactivation significantly impairs CML development and LSC burden in vivo and is upregulated independent of the Bcr-Abl1 kinase this receptor represents an attractive novel target. DisclosuresBruemmendorf:Novartis: Research Funding. Koschmieder:Roche: Other: Clinical Trial participation; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding.

Innate Immune Signal Pathway Activation Induced By Combined Loss of Del(5q) MDS Genes, Tifab and Mir-146a, Accelerates Hematopoietic Stem Cell Exhaustion Driven By Chronic Exposure to a TLR Ligands

Deletion of the long arm of chromosome 5 (del(5q)) is the most frequently observed chromosomal abnormality in patients with myelodysplastic syndrome (MDS). We previously reported that deletion of TRAF-interacting protein with forkhead-associated domain B (TIFAB) and miR-146a, two haploinsufficient genes in del(5q) MDS, activates the Toll-like receptor (TLR) signaling cascade in hematopoietic stem/progenitor cells (HSPC) by increasing TRAF6 protein stability and mRNA translation, respectively (Varney et al., JEM 2015 and Varney et al., Leukemia 2017). To investigate the epistasis of TIFAB and miR-146a, we generated a mouse model in which Tifab and miR-146a were simultaneously deleted (Tifab-/-; miR-146a-/-, herein dKO). Consistent with increased TRAF6 expression, dKO mice exhibit phosphorylation of IRAK1 and IRAK4 kinases, indicating activation of the TLR/innate immune pathway in bone marrow (BM) HSPC of dKO mice. To determine whether combined deletion of Tifab and miR-146a exaggerates the hematopoietic-specific defects observed in either Tifab- or miR-146a-deficient mice in vivo, BM cells from WT, Tifab-/-, miR-146a-/-, and dKO mice were transplanted into lethally-irradiated syngeneic recipient mice. As previously reported, a minor subset of Tifab-/- and ~ 50% of miR-146a-/- mice succumbed to a bone marrow failure (BMF)-like disease. For dKO mice, 80% of mice developed a more rapid and penetrant BMF-like disease as compared to loss of Tifab or miR-146a alone. Moribund dKO mice presented with leukopenia, severe anemia, and thrombocytopenia. Since TLR activation is a common feature of MDS due to overexpression and secretion of damage-associated molecular patterns (DAMPs) within the BM microenvironment (Monlish et al., Front Immunol 2016), we explored the biological effect of systemic TLR activation on hematopoietic stem cell (HSC) function of dKO mice following exposure to chronic low dose lipopolysaccharide (LPS), a TLR4 agonist. After 4 weeks, the proportion of myeloid cells in peripheral blood (PB) of LPS-treated dKO mice was significantly increased as compared to WT mice exposed to LPS. BM cellularity of dKO mice was dramatically decreased by LPS exposure while that of WT mice was only slightly affected by LPS. The proportion of CD48-CD150+Lineage-Sca1+c-Kit+ (LSK) of dKO mice was significantly reduced by exposure to LPS as compared to WT mice, which correlated with loss of HSC quiescence. We next performed competitive repopulation assays using WT and dKO BM cells exposed to LPS. As expected, the chimerism of donor-derived PB cells from LPS-treated WT mice was significantly lower than that from WT control mice. Intriguingly, LPS-treated dKO mice showed significantly reduced chimerism of donor-derived PB and CD48-CD150+ LSK BM cells as compared to vehicle-treated dKO mice or WT mice exposed to LPS. Importantly, the impaired hematopoietic reconstitution was more pronounced following serial BM transplantation. These results indicate that deletion of Tifab and miR-146a diminishes the long-term repopulating potential of HSC driven by in vivo TLR stimulation. To uncover the molecular consequences that explain the functional HSC defect of dKO mice exposed to TLR activation, RNA-sequencing was performed on LSK from dKO and WT mice treated with LPS. Following TLR activation, we observed differential expression of distinct genes in dKO LSK as compared to WT LSK. Pathway network analyses revealed changes in chromatin organization, epigenetic regulations, and cellular senescence in WT LSK exposed to LPS as compared to control WT cells. In contrast, dKO LSK exposed to LPS exhibited enrichment in pathways associated with axon guidance, carbohydrate metabolism, and megakaryocyte development and platelet production as compared to control dKO cells. These results suggest the differential impact of TLR stimulation on hematopoiesis in Tifab;miR-146a-deficient mice. Collectively, our findings indicate that innate immune signal pathway activation induced by combined loss of Tifab and miR-146a accelerates HSC exhaustion driven by chronic exposure to TLR ligands via the activation of distinct signal pathways. These findings have important implications in the role of TLR stimulation in the cellular consequence of MDS HSC. DisclosuresNo relevant conflicts of interest to declare.

Gain-of-Function of ASXL1 Truncating Protein in the Pathogenesis of Myeloid Malignancies

Introduction: Additional Sex Combs-Like 1 (ASXL1) is mutated at a high frequency in all forms of myeloid malignancies associated with poor prognosis. We and others have reported that loss of ASXL1 impairs hematopoietic stem/progenitor cell functions (HSC/HPCs) and leads to myeloid dysplastic syndrome (MDS) in mice. It remains controversial whether truncating mutations in ASXL1 result in gain- or loss-of-function, or whether they confer dominant-negative activity in vivo . Answering this question is critical for determining how to target these mutations therapeutically.Methods: We generated a transgenic mouse model using HS321/45-vav vector that expresses the Asxl1 Y588X cDNA under the control of the Vav1 promoter. The transgene contains the entire coding region of mouse Asxl1 (full-length) with a stop codon mutation at the Y588 site (Asxl1 Y588X), leading to a production of truncating protein ASXL1aa1-587 in the hematopoietic system only. Utilizing the transgenic model, we performed a serial hematopoietic analyses, including peripheral blood (PB) counts, histological characterization on blood smear, tissue sections of femur and spleen, and flow cytometric analysis. Bone marrow(BM) transplantation was performed to identify the effects of truncating ASXL1aa1-587 on HSC/HPCs functions. RNA-seq and ATAC-seq analyses were performed to survey the differentially expressed genes in HSC/HPCs of WT and Asxl1 Y588X Tg mice. Liquid chromatography-tandem mass spectrometry (LC-MS) and co-immunoprecipitation (IP) experiments were performed to identify the specific protein binding partner of ASXL1aa1-587. An epigenetic drug screening was used to identify specific targets to block the gain-of-function protein, ASXL1aa1-587, in the pathogenesis of myeloid malignancies in vivo.Results: The Asxl1Y588X Tg mice had an enlarged hematopoietic stem cell (HSC) pool, shortened survival, and predisposition to a spectrum of myeloid malignancies, thereby recapitulating the characteristics of myeloid malignancy patientswith ASXL1 mutations. Competitive transplantation of WT and Asxl1Y588XTg BM cells revealed that expression of A SXL 1 aa587 was associated with a higher percentage of CD45.2+ cells in the PB of the recipients, indicating that A SXL11-587aa expression increases HSC self-renewal capacity in vitro and in vivo. ATAC- and RNA-Seq analyses revealed that the ASXL11-587aa truncating protein expression results in more open chromatin in cKit+ cells compared to wildtype cells, accompanied by dysregulated expression of genes critical for HSC self-renewal and differentiation. LC-MS) and co-IP experiments showed that ASXL11-587aa acquired an interaction with BRD4. An epigenetic drug screening demonstrated a hyper-sensitivity of Asxl1Y588X Tg bone marrow cells to BET bromodomain inhibitors.Conclusion: This study demonstrates that ASXL11-587aa plays a gain-of-function role in promoting myeloid malignancies and that BET bromodomain inhibitors significantly reduced the colony forming capacity of ASXL1Y588XTgHSPCs. Our model provides a powerful platform to test therapeutic approaches of targeting the ASXL1 truncation mutations in myeloid malignancies. DisclosuresNo relevant conflicts of interest to declare.

Signal-Transducing Adaptor Protein-2 Blocks B Cell Recovery Under Hematological Stress at Pre-B Stage Via TLR4 Signaling

All lineages of blood cell production from hematopoietic stem cells (HSC) are maintained for an entire lifetime. That is consistent under homeostatic condition, while hematopoiesis is changed in response to various types of stress, including infections and aging. It has been reported that hematopoietic stem and progenitor cells (HSPC) are targets of pathogen products and danger signals. After the exposure to Gram-negative lipopolysaccharide (LPS) that is the ligand of Toll-like receptor (TLR) 4, HSC enter cell-cycle and differentiate into myeloid lineage cells while B lymphopoiesis is suppressed. Although the regulation of hematopoiesis by HSC is well studied, many questions still remain regarding how progenitors contribute the response to hematopoietic demands. Signal-transducing adaptor protein-2 (STAP-2) was cloned as a c-fms/M-CSFR interacting protein in 2003, and the interaction with a variety of signaling or transcriptional molecules such as STAT5, MyD88 and IκB kinase (IKK) in macrophages or T cells has been reported. The function as an adaptor protein in various cell types and signaling pathways, and its ubiquitous expression promoted us to investigate the effect of STAP-2 on HSPC under hematopoietic stress using gene-modified mice.We previously reported that hematopoiesis in STAP-2 knock out mice (KO) is normal under homeostatic condition. First in this study, competitive bone marrow (BM) transplantation experiment was conducted using KO. Four months after intravenous injection of Lineage- Sca1+ cKithigh (LSK) HSPC (CD45.2+) derived from KO or wild-type mice (WT) with competitor BM mononuclear cells (CD45.1+), the engraftment and lineage distribution were comparable, indicating that STAP-2 does not affect the function of long-term HSC. Interestingly, we found that chimerism in peripheral B cells at 1 month after transplantation in KO LSK transplanted mice was higher than control with statistical significance. In BM, STAP-2 deficiency increased pre-B and immature B cell cells, while there were no differences in numbers of LSK HSPC or Lin- Sca1+ cKitlow Flk2high common lymphoid progenitor (CLP) between WT and KO donor. To evaluate the role of STAP-2 in early B lymphopoiesis, we generated transgenic mice (Tg) that overexpress STAP-2 under the control of Eμ enhancer and Lck proximal promoter. The promoter could drive expression of the inserted cDNA in lymphoid lineage cells from CLP stage. In Tg, we observed significant decrease of B progenitors compared to WT BM. When HSPC (LSK or CLP) derived from Tg were cultured with OP9 stromal cells under B cell condition in the presence of IL-7, Flt3-ligand and SCF, the production of B220+ B cells were significantly suppressed. The same was true in stromal-cell free cultures. In colony-forming unit (CFU) assays, we found that STAP-2 overexpression decreased the ability to generate pre-B colonies, while the deficiency promoted high recovery with statistical significance (recovered colony number; 40.0 ± 1.4 in WT, 7.0 ± 1.4 in Tg, and 89.0 ± 5.7 in KO). To elucidate the detailed mechanisms, RNA-seq experiment with WT and Tg under steady-state as well as WT and KO 1 month after transplantation was conducted. Data among them were compared, and bioinformative approach with Ingenuity Pathway Analysis revealed that STAP-2 upregulates TLR4 signaling in pre-B cells during stress hematopoiesis. Consistent with the results, when LPS was added into CFU cultures, the recovered pre-B colony was suppressed. Moreover, deletion of STAP-2 partially cancelled the inhibitory effect of LPS on pre-B cell proliferation.In summary, we found that STAP-2 regulates the early B lymphopoiesis under hematopoietic stress. While STAP-2 did not influence stemness of HSC or homeostatic hematopoiesis, short-term recovery of B cells after transplantation was promoted by STAP-2 deficiency. This effect was induced by the activated proliferation of pre-B cells. Analyses with Tg mice confirmed the inhibitory effect of STAP-2 on early B lymphopoiesis. RNA-seq analyses have shown that activation of TLR4 signaling at pre-B stage occurs 1 month after transplantation as well as in Tg mice, and STAP-2 deficiency partially cancelled the inhibitory effects of LPS in CFU pre-B assays. Our study unveiled the critical role of lineage progenitors in short-term response to hematological demands and developmental stage-specific regulation of TLR4-STAP-2 pathway. DisclosuresOritani:Celgene: Speakers Bureau; Bristol-Myers Squibb: Research Funding, Speakers Bureau; MOCHIDA PHARMACEUTICAL: Speakers Bureau; Novartis Pharma: Consultancy, Speakers Bureau. Shibayama:Takeda Pharmaceutical Co.,LTD.: Honoraria, Research Funding; Fujimoto Pharmaceutical Co.: Honoraria, Research Funding; Jansen Pharmaceutical K.K.: Honoraria; Bristol-Meyer Squibb K.K.: Honoraria, Research Funding; Ono Pharmaceutical Co.,LTD.: Honoraria, Research Funding; Novartis Pharma K.K.: Honoraria, Research Funding; Mundipharma K.K.: Honoraria, Research Funding; Celgene K.K.: Honoraria, Research Funding. Kanakura:Alexion Pharmaceuticals, Inc.: Honoraria, Research Funding; Bristol Myers: Research Funding; Fujimotoseiyaku: Research Funding; Chugai Pharmaceutical: Research Funding; Astellas: Research Funding; Pfizer: Research Funding; Nippon Shinyaku: Research Funding; Toyama Chemical: Research Funding; Shionogi: Research Funding; Eisai: Research Funding; Kyowa Hakko Kirin: Research Funding.

Extracellular DEK Regulates Hematopoiesis through CXCR2-Mediated Intracellular Signaling That Does Not Require Its Translocation into the Nucleus or Binding to DNA

DEK is a highly conserved, ubiquitously expressed and structurally unique DNA-binding nuclear protein involved in transcription, DNA replication, and DNA repair implicating a role for DEK in gene regulation. Interestingly, hematopoietic cells, following stimulation with inflammatory mediators, can actively secrete DEK, which can then be taken up by cells via binding to heparan sulfate proteoglycans (HSPGs), translocating to the nucleus, and mediating its normal function of gene regulation. Previous work from our group indicates that both endogenous, intracellular DEK and secreted, extracellular DEK regulates normal hematopoiesis by decreasing numbers of hematopoietic progenitor cells (HPCs) and increasing numbers of hematopoietic stem cells (HSCs) suggesting that DEK plays a role in regulating HSC/HPC function and/or cell fate. Extracellular DEK-mediated regulation of hematopoiesis occurs in a HSPG-, CXCR2-, Gai protein-dependent manner. Neutralizing these pathways inhibits extracellular DEK regulation of HSCs and HPCs. However, what was not known is whether the HSPG-dependent internalization of DEK is required for the extracellular DEK-mediated regulation of hematopoiesis as HSPGs are also required for maximum binding efficiency of ELR-CXC chemokines to CXCR2. To address this unknown, two mutant forms of recombinant (r)DEK were utilized: DEK that lacks its nuclear translocation sequence (NTS) and DEK that lacks its DNA-binding motif (DBM). First we examined whether NTS- or DMB-rDEK could alter HPC or HSC numbers in vitro / ex vivo . We had previously shown that C57Bl/6 bone marrow (BM) cells when in culture with rDEK inhibited HPC colony formation in a dose-dependent manner, with the minimum dose of WT rDEK required for inhibition being 12.5 nM (p=0.002). Both NTS- and DMB-rDEK also inhibited HPC colony formation in a dose-dependent manner starting at 12.5 nM (p=0.03 and p=0.02 respectively) suggesting that neither the ability of DEK to get into the nucleus or bind to DNA is required to regulate HPC numbers in BM. To examine whether NTS- or DMB-rDEK can still enhance HSC numbers, ex vivo HSC expansion assays were performed. C57Bl/6 lineage negative BM cells were cultured in SCF, TPO, and Flt3L with vehicle control, WT-rDEK, NTS-rDEK, or DMB-rDEK then examined for HSC expansion on days 4 and 7. On both days, WT-, NTS-rDEK and DMB-rDEK significantly expanded long-term HSC numbers when compared to vehicle control, suggesting that like DEK-mediated suppression of HPCs, DEK-mediated expansion of HSCs does not require DEK to translocate to the nucleus or bind to DNA. To examine whether extracellular NTS- and DMB-rDEK can still mediate one of DEK's known nuclear functions, heterochromatin stability, DEK-/- lineage negative BM cells were incubated for 12 hours with WT rDEK, NTS-rDEK or DMB-rDEK then examined for H3K9 trimethylation in HSCs and HPCs by ImageStream flow cytometry. Neither NTS- or DMB-rDEK were able to enhance the trimethylation of H3K9 in DEK-/- HSCs or HPCs. However, incubating DEK-/- lineage negative BM with WT rDEK, increased H3K9me3 in the nucleus of the cells by ~26%. When utilizing WT BM, incubating cells for 12 hours with WT, NTS- or DMB-rDEK did not result in changes in H3K9Me3 that was detectable by ImageStream technology suggesting that extracellular DEK-mediated changes in chromatin remodeling in HSCs and HPCs only occurs when the cells lack endogenous DEK. Co-culturing C57Bl/6 BM cells with WT rDEK mediated AKT and ERK1/2 phosphorylation in HSCs and ERK1/2 in HPCs which is blocked when BM cells are pretreated with a neutralizing antibody against CXCR2 or when preteated with heparin neutralizing HSPGs. Both NTS- and DMB-rDEK were able to mediate similar activation of AKT and ERK1/2 in HSCs and HPCs as WT rDEK but not when the BM was pretreated with anti-CXCR2 antibody or heparin. Taken together, these data suggest that extracellular DEK functions to regulate HSC and HPC numbers/function by binding to CXCR2 to mediate intracellular signaling events in C57Bl/6 BM cells. The fact that HSPGs are required for extracellular DEK's effect, but DEK does not have to get into the nucleus and bind DNA, also suggests that internalization of DEK is not required to regulate hematopoiesis in cells with normal levels of endogenous DEK. Therefore, like the ELR-CXC chemokines, DEK requires HSPGs for its CXCR2-mediated signaling processes in both HSCs and HPCs. DisclosuresNo relevant conflicts of interest to declare.

CD40 Activity on Mesenchymal Cells Negatively Regulates OX40L to Maintain Bone Marrow Immune Homeostasis Under Stress Conditions.

BackgroundWithin the bone marrow (BM), mature T cells are maintained under homeostatic conditions to facilitate proper hematopoietic development. This homeostasis depends upon a peculiar elevated frequency of regulatory T cells (Tregs) and immune regulatory activities from BM-mesenchymal stem cells (BM-MSCs). In response to BM transplantation (BMT), the conditioning regimen exposes the BM to a dramatic induction of inflammatory cytokines and causes an unbalanced T-effector (Teff) and Treg ratio. This imbalance negatively impacts hematopoiesis, particularly in regard to B-cell lymphopoiesis that requires an intact cross-talk between BM-MSCs and Tregs. The mechanisms underlying the ability of BM-MSCs to restore Treg homeostasis and proper B-cell development are currently unknown.MethodsWe studied the role of host radio-resistant cell-derived CD40 in restoring Teff/Treg homeostasis and proper B-cell development in a murine model of BMT. We characterized the host cellular source of CD40 and performed radiation chimera analyses by transplanting WT or Cd40-KO with WT BM in the presence of T-reg and co-infusing WT or - Cd40-KO BM-MSCs. Residual host and donor T cell expansion and activation (cytokine production) and also the expression of Treg fitness markers and conversion to Th17 were analyzed. The presence of Cd40+ BM-MSCs was analyzed in a human setting in correlation with the frequency of B-cell precursors in patients who underwent HSCT and variably developed acute graft-versus-host (aGVDH) disease.ResultsCD40 expression is nearly undetectable in the BM, yet a Cd40-KO recipient of WT donor chimera exhibited impaired B-cell lymphopoiesis and Treg development. Lethal irradiation promotes CD40 and OX40L expression in radio-resistant BM-MSCs through the induction of pro-inflammatory cytokines. OX40L favors Teff expansion and activation at the expense of Tregs; however, the expression of CD40 dampens OX40L expression and restores Treg homeostasis, thus facilitating proper B-cell development. Indeed, in contrast to dendritic cells in secondary lymphoid organs that require CD40 triggers to express OX40L, BM-MSCs require CD40 to inhibit OX40L expression.ConclusionsCD40+ BM-MSCs are immune regulatory elements within BM. Loss of CD40 results in uncontrolled T cell activation due to a reduced number of Tregs, and B-cell development is consequently impaired. GVHD provides an example of how a loss of CD40+ BM-MSCs and a reduction in B-cell precursors may occur in a human setting.

Impact of Galectin-3 tissue deletion in renal damage and type-3 cardio-renal syndrome

We have recently reported the crucial role of Galectin-3 (Gal3) in the progression of type 3 cardio-renal syndrome (CRS-3). Indeed, Gal3 overexpression after renal ischemia reperfusion (IRr) was associated with cardiac fibrosis and dysfunction, which were prevented by Gal3 pharmacological or genetic inhibition. Our present aim was to better define the initial role of kidney Gal3 for triggering CRS-3. A murine irradiated and graft model having IRr was used to differentiate the Gal3 expressed by tissue (WT irradiated mice grafted with Gal3-/- bone marrow (BM) cells, WT Gal3-/-B M) from Gal3 expressed by immune cells (Gal3-/- irradiated mice grafted with WT BM cells: Gal3-/- WTB M) and were killed after 2 or 28 days. Cardiac and renal phenotypes were evaluated using echocardiography and real-time PCR. After IRr, WT Gal3-/-B M exhibit normal cardiac phenotype whereas Gal3-/- WTB M developed cardiac dysfunction and high Fibronectin (Fn) mRNA expression ( Fig. 1 A). In contrast, WT Gal3-/-B M showed short- and long-term kidney damages with increased renal NGAL mRNA level by day 2 and increased Fn mRNA by 28d ( Fig. 1 B). In kidney after IRr, the increased levels of endothelial marker transcripts (ICAM-1, VCAM-1) mainly in WT Gal3-/-B M mice suggested that tissue Gal3 expression is involved in the endothelial activation. After IRr the organ dysfunction varies according to the Gal3 cell type origin; kidney Gal3 plays a major role in renal damage, whereas Gal3 from infiltrated BM cells in the heart is deleterious for cardiac function. It remains now to define how tissue Gal3 acts and interacts with endothelial cells, to assess Gal3 as a key-point for CRS-3 understanding.

PI3KC2β inactivation stabilizes VE-cadherin junctions and preserves vascular integrity.

Endothelium protection is critical, because of the impact of vascular leakage and edema on pathological conditions such as brain ischemia. Whereas deficiency of class II phosphoinositide 3-kinase alpha (PI3KC2α) results in an increase in vascular permeability, we uncover a crucial role of the beta isoform (PI3KC2β) in the loss of endothelial barrier integrity following injury. Here, we studied the role of PI3KC2β in endothelial permeability and endosomal trafficking in vitro and in vivo in ischemic stroke. Mice with inactive PI3KC2β showed protection against vascular permeability, edema, cerebral infarction, and deleterious inflammatory response. Loss of PI3KC2β in human cerebral microvascular endothelial cells stabilized homotypic cell-cell junctions by increasing Rab11-dependent VE-cadherin recycling. These results identify PI3KC2β as a potential new therapeutic target to prevent aggravating lesions following ischemic stroke.

Hdac3 regulates bone modeling by suppressing osteoclast responsiveness to RANKL

Hdac3 is a lysine deacetylase that removes acetyl groups from histones and additional proteins. Although Hdac3 functions within mesenchymal lineage skeletal cells are defined, little is known about Hdac3 activities in bone-resorbing osteoclasts. In this study we conditionally deleted Hdac3 within Ctsk-expressing cells and examined the effects on bone modeling and osteoclast differentiation in mice. Hdac3 deficiency reduced femur and tibia periosteal circumference and increased cortical periosteal osteoclast number. Trabecular bone was likewise reduced and was accompanied by increased osteoclast number per trabecular bone surface. We previously showed that Hdac3 deacetylates the p65 subunit of the NF-κB transcriptional complex to decrease DNA-binding and transcriptional activity. Hdac3-deficient osteoclasts demonstrate increased K310 NF-κB acetylation and NF-κB transcriptional activity. Hdac3-deficient osteoclast lineage cells were hyper-responsive to RANKL and showed elevated ex vivo osteoclast number and size and enhanced bone resorption in pit formation assays. Osteoclast-directed Hdac3 deficiency decreased cortical and trabecular bone mass parameters, suggesting that Hdac3 regulates coupling of bone resorption and bone formation. We surveyed a panel of osteoclast-derived coupling factors and found that Hdac3 suppression diminished sphingosine-1-phosphate production. Osteoclast-derived sphingosine-1-phosphate acts in paracrine to promote bone mineralization. Mineralization of WT bone marrow stromal cells cultured with conditioned medium from Hdac3-deficient osteoclasts was markedly reduced. Expression of alkaline phosphatase, type 1a1 collagen, and osteocalcin was also suppressed, but no change in Runx2 expression was observed. Our results demonstrate that Hdac3 controls bone modeling by suppressing osteoclast lineage cell responsiveness to RANKL and coupling to bone formation.

PHF6 Restricts AML Acceleration By Promoting Myeloid Differentiation Genes in Leukemic Cells

Acute myeloid leukemia is caused by the accumulation of mutations in hematopoietic stem and myeloid progenitor cells, resulting in increased self-renewal, inhibition of differentiation, and aberrant proliferation. Although genomic studies have comprehensively identified genes that are mutated in acute leukemias, the functional roles of many of them, and the consequences of their mutations, remain poorly understood. PHF6 (PHD-finger protein 6) is an X-linked gene that is mutated in 3.2% of de novo AML, 4.7% CMML, 3% MDS, and 1.6% CML patients. Two-thirds of somatic mutations in PHF6 are frameshift and nonsense mutations distributed throughout the gene body, resulting in loss of PHF6 protein. One-third of the mutations are point mutations clustered in the ePHD2 (extended PHD) domain, and the consequence of these mutations on PHF6 function is unknown. The functional role of PHF6 and the mechanism by which PHF6 mutations accelerate AML has not yet been determined. In this study, we delineate the cellular and molecular function of PHF6 in AML using in vitro and in vivo models. In agreement with recently published reports, we found that pan-hematopoietic deletion of Phf6 using the Vav-Cre recombinase system gave competitive transplantation advantage to HSCs, with sustained multi-lineage reconstitution without exhaustion over three rounds of serial transplantations, demonstrating that Phf6 represses HSC self-renewal. However, loss of Phf6 alone was insufficient to cause hematopoietic malignancy in the mouse model when monitored for one year. To determine the function of PHF6 in AML progression, we transduced cKO (Vav-Cre; Phf6 flox) or WT (Vav-Cre only) bone marrow cells with MSCV retrovirus expressing HOXA9 (WT+HOXA9 and cKO+HOXA9), and transplanted into irradiated recipient mice. The resulting HOXA9-driven AML was greatly accelerated in the Phf6 cKO background, with recipient mice succumbing faster (median survival 119 days) as compared to recipients transduced with HOXA9-transduced WT cells (median survival &amp;gt;180 days, p=0.003) (Fig 1A). This was also reflected by an increase in the number of circulating immature leukemic cells in peripheral blood at earlier timepoints. HOXA9-transduced cKO cells showed higher serial replating ability in an in vitro colony forming assay as compared with HOXA9-transduced WT cells (Fig 1B). We further investigated the molecular function of PHF6 using the THP-1 human AML cell line. PHF6 is a chromatin-binding protein with two ePHD domains, and its binding partners and pattern of chromatin occupancy are unclear. Using ChIP-Seq, we identified that PHF6 occupies enhancers, and its peaks show striking alignment with the peaks of the key myeloid transcription factors (TFs) RUNX1, PU.1, and IRF8 (Fig 1C). To assess the effect of the clinically relevant point mutation R274Q (in the ePHD2 domain) on the transcriptional effects produced by PHF6, we first generated a PHF6 KO clone from the THP-1 cell line, and then re-expressed either WT PHF6 or R274Q-mutant PHF6 in this KO line. Re-expression of WT PHF6 rescued the extensive gene expression changes produced by its knockout, but R274Q-mutant PHF6 was unable to produce any gene expression changes, indicating that it is a "transcriptionally dead" mutant (Fig 1D). Gene Ontology analysis of transcriptome changes induced by WT PHF6 showed that PHF6 promotes the expression of myeloid differentiation gene sets. In summary, PHF6 restricts AML progression by binding enhancers with key myeloid TFs, and promoting the expression of myeloid differentiation genes. R274Q mutation renders PHF6 unable to exert any downstream expression changes, indicating that the ePHD2 domain (where R274 is located, clustered with other amino acids showing point mutations in hematopoietic malignancies) is critical for PHF6 function, and likely mediates important functional interactions with chromatin partners. Our future work will involve dissection of the sequence of molecular events governed by PHF6 following enhancer occupancy, and the role of PHF6 in repressing AML self-renewal and promoting differentiation. Disclosures No relevant conflicts of interest to declare.

Hypoxia-induced miR-210 modulates inflammatory response and fibrosis upon acute peripheral ischemia

Abstract Introduction We previously demonstrated in mouse models of peripheral and heart ischemia that miR-210 is necessary and sufficient to stimulate blood perfusion recovery, as well as arteriolar and capillary density increase following ischemic damage. Aim To clarify the role of inflammatory cells in miR-210-induced angiogenesis. Methods and results In a mouse model of acute limb ischemia, miR-210 loss-of-function was obtained by administration of complementary anti-miR-210 LNA oligonucleotides (anti-210), while doxycycline-inducible miR-210 transgenic mice (Tg210) were used for gain-of-function experiments. Transcriptomic and gene ontology analysis in ischemic gastrocnemius muscles upon miR-210 blocking displayed an enrichment of categories related not only to angiogenesis, but also to inflammation, suggesting that miR-210 decrease prompted a pro-inflammatory and anti-regenerative response. Accordingly, immunofluorescence staining of ischemic muscles of anti-210 treated mice, showed an increased presence of granulocytes (Scr = 28±7, anti-210 = 108±17 cells/mm2, p&amp;lt;0.001), T and B lymphocytes (Scr = 32±8 SE, anti-210 = 112±19 cells/mm2, p&amp;lt;0.003; Scr = 45±10, anti-210 = 103±14 cells/mm2, p&amp;lt;0.006, respectively) and macrophages (Scr = 17±1, anti-210 = 27±4 cells/mm2; p&amp;lt;0.03), with a higher M1/M2 macrophage ratio (Scr = 0.6±0.1, anti-210 = 1.7±0.3; p&amp;lt;0.02). Conversely macrophages (WT = 17±2, Tg210 = 5±1 cells/mm2, p&amp;lt;0.003) and M1/M2 ratio (WT = 1.0±0.1, Tg210 = 0.3±0.1, p&amp;lt;0.02) were decreased in ischemic TG-210 mice. To clarify the role of inflammatory cells in miR-210-induced angiogenesis, bone-marrow (BM) transplantation experiments were performed. Tg210 mice transplanted with WT BM cells (BM-wt/R-Tg210), compared with WT mice transplanted with Tg210 BM (BM-Tg210/R-wt) showed increased blood perfusion (vascularity ratio: BM-wt/R-Tg210 = 0.8±0.1, BM-Tg210/R-wt = 0.6±0.1; p&amp;lt;0.01) and capillary density after ischemia (BM-wt/R-Tg210 = 497±41, BM-Tg210/R-wt = 212±32 cap./mm2; P&amp;lt;0.00001). Thus, miR-210 expression in the muscle and not in the BM-derived cells was crucial for miR-210-stimulated angiogenesis. Interestingly, BM-wt/R-Tg210 mice also showed increased fibrotic areas (sirius red staining: BM-wt/R-Tg210 = 12±2, BM-Tg210/R-wt = 22±3 A.U.; p&amp;lt;0.01), characterized by α-SMA+, vimentin+ and collagen V+ stainings. Fibrotic regions were enriched in cells double-positive for both CD206/α-SMA and CD45/α-SMA, as well as in phospho-Smad3+ cells, suggesting the activation of the TGFβ pathway. In vitro experiments showed higher expression of α-SMA and collagens in TG-210 BM-derived macrophages compared to WT, both in the presence and in the absence of TGFβ (α-SMA: w/o TGFβ 2.3±1.4 fold increase p&amp;lt;0.004; TGFβ+ 11.3±2 fold increase p&amp;lt;0.003). Conclusions Collectively, these data show that a miR-210 enriched milieu was sufficient to improve angiogenesis after ischemia. Moreover, a context-dependent regulation by miR-210 of the inflammatory response and of fibrosis were identified. Funding Acknowledgement Type of funding source: Public hospital(s). Main funding source(s): Italian Ministry of health: Ricerca Corrente and 5X1000

3132 – IMPACT OF THE INFLAMED BONE MARROW NICHE ON THE PROGRESSION OF MYELOPROLIFERATIVE NEOPLASIA

Bcr/Abl-negative myeloproliferative neoplasia (MPN) is characterized by driver mutations in MPL, CALR, or JAK2, as well as an inflammatory microenvironment. The acquisition of clonal abnormalities and secondary mutations in MPN patients can progress the disease to Acute Myeloid Leukemia (AML), resulting in bone marrow (BM) fibrosis and BM failure. For these patients, treatment options fall short and do not include approaches to prevent leukemic transformation. Our group has developed an animal model for inflammation where loss of RBPJ/Notch signaling in the BM niche leads to upregulation of miR155 and an accumulation of proinflammatory cytokines. We propose to use this model (RBPJKO mouse) to study the influence of chronic inflammation on JAK2V617F-driven MPN progression. We hypothesize that chronic inflammation in the BM niche: i) promotes proliferation and genomic instability in hematopoietic cells; and ii) cooperates with preexisting genetic mutations such as JAK2V617F to promote clonal expansion and selection, accelerating disease progression. Our preliminary studies indicate that JAK2V617F hematopoietic cells drive a more aggressive disease when exposed to chronic inflammation following transplant into RBPJKO inflamed BM niches. Indeed, JAK2V617F/RBPJKO recipients exhibited accelerated neutrophilia, higher myeloid skewing and increased bone marrow cellularity and splenomegaly, when compared to JAK2V617F/RBPJWT recipients. By using alkaline comet assay and gH2Ax foci, we also demonstrate that JAK2V617F cooperates with the inflamed BM niche to promote more DNA single- and double-strand breaks in Lin- progenitors in vivo. To further study these events, we optimized an in vitro model in which WT and JAK2V617F BM Lin- progenitors are co-cultured with RBPJKO or RBPJWT BM-derived stroma. JAK2V617F cells showed increased proliferation, mitochondrial ROS accumulation, elevated 53BP1 foci and increased γH2AX MFI when exposed to the inflammatory conditions supported by RBPJKO stroma. Taken together, these data suggest that chronic inflammation can accentuate the MPN phenotype, promote replicative stress and lead to buildup of DNA-damaging ROS in JAK2V617F MPN cells. This work has the potential to provide us with new insights into the mechanisms of MPN progression and the opportunity to identify new therapeutic approaches.

Early B cell protein λ5 affects skeletal homeostasis

Abstract There are strong indications that B lineage cells play a key role in bone homeostasis. The preB cell receptor is composed of an immunoglobulin mu H chain, VpreB and immunoglobulin λ5. Young λ5 deficient mice have reduced numbers of preB cells and mature B cells, moreover; their bone appears to be normal. With time, they achieve normal numbers of mature B cells while exhibiting major bone loss. To test whether expression of λ5 is playing a critical role maintaining bone homeostasis, we evaluated bone in: mice lacking immunoglobulin JH (JH −/− λ5+/+). These mice lack both preB and mature B cells, but maintain expression of λ5 in progenitor B cells. Mice deficient in both JH and λ5 (JH −/− λ5−/−) have no preB or mature B cells, and also no λ5. Using both μCT and histologic evaluation, we compared femur and skull at six months of age in these three mutant strains to normal controls. JH −/− λ5+/+ femur and skull were similar to WT. Both JH +/+λ5−/− and JH −/− λ5−/− mice showed significant decrease in femoral trabecular bone and increased loss of skull bone. Irradiated WT mice transplanted with JH −/− λ5−/− bone marrow cells developed defective femoral and cranial bones. In contrast, JH −/−λ5−/− mice that received WT bone marrow cells recovered bone architecture. In vitro culture of bone progenitors from JH +/+λ5−/− and JH −/− λ5−/− mice showed decreased ability of bone formation. These data underscore the importance of immunoglobulin λ5 in skeletal development.