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Hematopoietic stem cells (HSCs) balance self-renewal and differentiation to maintain hematopoietic fitness throughout life. In steady-state conditions, HSC exhaustion is prevented by the maintenance of most HSCs in a quiescent state, with cells entering the cell cycle only occasionally. HSC quiescence is regulated by retinoid and fatty-acid ligands of transcriptional factors of the nuclear retinoid X receptor (RXR) family. Here, we show that dual deficiency for hematopoietic RXRa and RXRb induces HSC exhaustion, myeloid cell/megakaryocyte differentiation, and myeloproliferative-like disease. RXRa and RXRb maintain HSC quiescence, survival, and chromatin compaction; moreover, transcriptome changes in RXRa;RXRb-deficient HSCs include premature acquisition of an aging-like HSC signature, MYC pathway upregulation, and RNA intron retention. Fitness loss and associated RNA transcriptome and splicing alterations in RXRa;RXRb-deficient HSCs are prevented by Myc haploinsufficiency. Our study reveals the critical importance of RXRs for the maintenance of HSC fitness and their protection from premature aging. We thank the members of the J.A.C. and M.R. laboratories for extensive discussions and critiques of the manuscript. We thank Daniel Metzger (Université de Strasbourg, France) for Rxrbf/f 418 mice, Juan Carlos Zúñiga-Pflücker (Sunnybrook Health Sciences Centre, Canada) for OP9-NL1 cells, Daniel Jiménez-Carretero (CNIC) for t-SNE analysis, the CRG (Barcelona, Spain) Genomics Unit for ATACseq sequencing, and S. Bartlett (CNIC) for editorial assistance. We also thank the staff of the CNIC Cellomics and Animal facilities for technical support. This study was supported by grants from the Spanish Ministerio de Ciencia e Innovación (MICIN) (SAF2017-90604-REDT-NurCaMein, RTI2018- 095928-B100, and PID2021-122552OB-I00), La Marató de TV3 Foundation (201605-32), and the Comunidad de Madrid (MOIR-B2017/BMD-3684) to M.R and from the Formación de Profesorado Universitario (FPU17/01731) program (MICIN) to J.P. The project also received funding from the US National Institutes of Health (R01 DK124115, P01 HL158688, R01 HL147536, R01 CA237016 and U54 DK126108 to J.A.C). The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovación (MCIN), and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence (grant CEX2020-001041-S funded by MICIN/AEI/10.13039/501100011033). Sí

The orphan nuclear receptor Nur77 is an immediate-early response gene that based on tissue and cell context is implicated in a plethora of cellular processes, including proliferation, differentiation, apoptosis, metabolism, and inflammation. Nur77 has a ligand-binding pocket that is obstructed by hydrophobic side groups. Naturally occurring, cell-endogenous ligands have not been identified, and Nur77 transcriptional activity is thought to be regulated through posttranslational modification and modulation of protein levels. To determine whether Nur77 is transcriptionally active in hematopoietic cells in vivo, we used an upstream activating sequence (UAS)-GFP transgenic reporter. We found that Nur77 is transcriptionally inactive in vivo in hematopoietic cells under basal conditions, but that activation occurs following cytokine exposure by G-CSF or IL-3. We also identified a series of serine residues required for cytokine-dependent transactivation of Nur77. Moreover, a kinase inhibitor library screen and proximity labeling-based mass spectrometry identified overlapping kinase pathways that physically interacted with Nur77 and whose inhibition abrogated cytokine-induced activation of Nur77. We determined that transcriptional activation of Nur77 by G-CSF or IL-3 requires functional JAK and mTor signaling since their inhibition leads to Nur77 transcriptional inactivation. Thus, intracellular cytokine signaling networks appear to regulate Nur77 transcriptional activity in mouse hematopoietic cells.

In this paper, we use difference-in-differences estimation to examine the effect of the Belt and Road Initiative on China's export potential to the countries along the Belt and Road routes. The results show that China's export potential to the Belt and Road countries rose significantly after the initiative began, especially for exports of products in capital intensive industries. We also distinguish between different regions and find a much stronger effect for countries in the Association of Southeast Asian Nations and West Asia.

The retinoid X receptor α (RXRA) has been implicated in diverse hematological processes. To identify natural ligands of RXRA that are present in hematopoietic cells, we adapted an upstream activation sequence-green fluorescent protein (UAS-GFP) reporter mouse to detect natural RXRA ligands in vivo. We observed reporter activity in diverse types of hematopoietic cells in vivo. Reporter activity increased during granulocyte colony-stimulating factor (G-CSF)-induced granulopoiesis and after phenylhydrazine (PHZ)-induced anemia, suggesting the presence of dynamically regulated natural RXRA ligands in hematopoietic cells. Mouse plasma activated Gal4-UAS reporter cells in vitro, and plasma from mice treated with G-CSF or PHZ recapitulated the patterns of reporter activation that we observed in vivo. Plasma from mice with dietary vitamin A deficiency only mildly reduced RXRA reporter activity, whereas plasma from mice on a fatty acid restriction diet reduced reporter activity, implicating fatty acids as plasma RXRA ligands. Through differential extraction coupled with mass spectrometry, we identified the long-chain fatty acid C24:5 as a natural RXRA ligand that was greatly increased in abundance in response to hematopoietic stress. Together, these data suggest that natural RXRA ligands are present and dynamically increased in abundance in mouse hematopoietic cells in vivo. We thank the Alvin J. Siteman Cancer Center at Washington University School of Medicine and Barnes-Jewish Hospital in St. Louis, MO. for the use of the Flow Cytometry Core. The Siteman Cancer Center is supported in part by an NCI Cancer Center Support Grant P30 CA91842. We thank High-Throughput Screening Center at Washington University School of Medicine in St. Louis, MO. We thank Deborah Laflamme for technical assistance and Feng Gao for statistical assistance. This work was supported by NIH R01 HL128447 (JS Welch), NIH P50 CA171963 (Project 1, JS Welch), and by grants from the Spanish Ministry of Economy and Competitiveness (SAF2015-64287R, SAF2015-71878-REDT) (M Ricote). The mass spectrometry facility at Washington University is supported by NIH P30 DK020579, Daniel Ory. J.S.W., H.N. and M.R. designed experiments, performed experiments, and wrote the manuscript. H.F., O.M., G.H., M.P.M, T.E.F., G.R.B. designed and performed experiments. Sí

Summary NtCOI1, a tobacco orthologue of the Arabidopsis jasmonate co-receptor COI1, was shown to regulate primary carbohydrate metabolism in both reproductive and vegetative organs of Nicotiana tabacum L. cv. TN90., Jasmonate (JA) plays an important role in regulating plant male fertility and secondary metabolism, but its role in regulating primary metabolism remains unclear. The F-box protein CORONATINE INSENSITIVE 1 (COI1) is a critical component of the JA receptor, and mediates JA-signalling by targeting JASMONATE ZIM-domain (JAZ) proteins for proteasomal degradation in response to JA perception. Here, we found that RNA interference-mediated knockdown of NtCOI1 in tobacco (Nicotiana tabacum L. cv. TN90) recapitulated many previously observed phenotypes in coi1 mutants, including male sterility, JA insensitivity, and loss of floral anthocyanin production. It also affected starch metabolism in the pollen, anther wall, and floral nectary, leading to pollen abortion and loss of floral nectar. Transcript levels of genes encoding starch metabolism enzymes were significantly altered in the pollen, anther wall, and floral nectary of NtCOI1-silenced tobacco. Changes in leaf primary metabolism were also observed in the NtCOI1-silenced tobacco. The expression of NtMYB305, an orthologue of MYB305 previously identified as a flavonoid metabolic regulator in Antirrhinum majus flowers and as a floral-nectar regulator mediating starch synthesis in ornamental tobacco, was extremely downregulated in NtCOI1-silenced tobacco. These findings suggest that NtCOI1 functions upstream of NtMYB305 and plays a fundamental role in coordinating plant primary carbohydrate metabolism and correlative physiological processes.

Retinoid therapy transformed response and survival outcomes in acute promyelocytic leukemia (APL), but has demonstrated only modest activity in non-APL forms of acute myeloid leukemia (AML). The retinoic acid receptors (RARs) and retinoid X receptors (RXRs) are ligand-activated transcription factors that influence hematopoietic stem cell self-renewal and differentiation. In normal hematopoiesis, RARA and RXRA are dynamically regulated during myeloid maturation, with highest expression in mature neutrophils. In acute myeloid leukemia (AML) RARA and RXRA have parallel expression among AML subtypes, with the highest expression in M4/M5 myelomonocytic and monocytic subtypes. Using a murine reporter assay, we found that natural RXRA ligands, but not RARA ligands, were present in vivo in primary mouse myelomonocytic leukemia cells (derived with an MLL-AF9 retrovirus) and acted as tumor suppressors. RXR ligands were absent in erythroleukemia (derived with TLS-ERG) or T-cell leukemia (derived with activated Notch1), suggesting the presence of natural RXRA ligands specifically in myelomonocytic leukemia. Moreover, we found that deletion of Rxra and Rxrb was naturally selected in MLL-AF9, but not in Notch1 or TLS-ERG leukemias, and that loss of Rxra and Rxrb accelerated leukemic growth. This suggests that Rxrs act as tumor suppressors, but only when exposed to natural ligands. In MLL-AF9 derived leukemia cells, pharmacologic treatment with single-agent retinoid led to modest growth inhibition due to non-permissive activity of the RARA:RXR heterodimer, whereas concurrent activation of both RARA, with all-trans retinoic acid (a pan-RAR ligand), and RXR, by bexarotene (a pan-RXR ligand), enabled efficient co-repressor release and synergistic leukemic apoptosis. We observed that co-repressors release (SMRT/NCoR) from the RARA:RXRA heterodimer was specifically associated with RARA activation, whereas growth inhibition required RXR binding, and this occurred through apoptosis rather than maturation. Generating a series of RXRA mutant proteins we demonstrated the active contribution of RXRA to the activity of the RARA:RXRA heterodimer, specifically requiring the activation domains (AF1 and AF2) and the ability to recruit co-activator to the RXRA element. Finally, we observed a significant dose-dependent effect of combination ATRA and bexarotene treatment in in vivo in MLL-AF9 leukemic mice with a striking reduction in the tumor burden of treated mice compared to the control cohort. These data provide a strategy for clinical retinoid therapies in leukemias beyond acute promyelocytic leukemia and provide a mechanism for integrating retinoid therapy into future clinical trials. Disclosures No relevant conflicts of interest to declare.

Nicotine is a secondary metabolite that is important to the defense system and commercial quality of tobacco (Nicotiana tabacum L.). Jasmonate and its derivatives (JAs) are phytohormone regulators of nicotine formation; however, the underlying molecular mechanism of this process remains largely unclear. Owing to the amphitetraploid origin of N. tabacum, research on screening and identification of nicotine-synthetic mutants is relatively scarce. Here, we describe a method based on JA-sensitivity for screening nicotine mutants from an activation-tagged population of tobacco. In this approach, the mutants were first screened for abnormal JA responses in seed germination and root elongation, and then the levels of nicotine synthesis and expression of nicotine synthetic genes in the mutants with altered JA-response were measured to determine the nicotine-synthetic mutants. We successfully obtained five mutants that maintained stable nicotine contents and JA responses for three generations. This method is simple, effective and low-cost, and the finding of transcriptional changes of nicotine synthetic genes in the mutants shows potentials for identifying novel regulators involved in JA-regulated nicotine biosynthesis.

SummaryThough antiplatelet drugs are proven beneficial to patients with coronary heart disease and stroke, more effective and safer antiplatelet drugs are still needed. In this study we report the antiplatelet effects and mechanism of BF0801, a novel adenine derivative. BF0801 dramatically inhibited platelet aggregation and ATP release induced by ADP, 2MeSADP, AYPGKF, SFLLRN or convulxin without affecting shape change in vitro. It also potentiated the inhibitory effects of adenosine-based P2Y12 antagonist AR-C69931MX or phosphodiesterase (PDE) inhibitor IBMX on platelet aggregation. The cAMP levels in both resting and forskolin-stimulated platelets were increased by BF0801 suggesting its PDE inhibitor activity, which is further confirmed by the concentration-dependent suppression of BF0801 on the native and recombinant PDE. Similar to AR-C69931MX, BF0801 drastically inhibited 2MeSADP-induced adenylyl cyclase inhibition in platelets indicating its P2Y12 antagonism activity, which is substantiated by the inhibition of BF0801 on the interaction between ADP and P2Y12 receptor expressed in CHO-K1 cells measured by atomic force microscopy. Moreover, we confirmed the antiplatelet effects of BF0801 using platelets from rats intravenously given BF0801. In summary, for the first time we developed a novel adenine derivative bearing dual activities of PDE inhibition and P2Y12 antagonism, which may have therapeutic advantage as a potential antithrombotic drug.

Mushroom-like ZnO microcrystals have been prepared via a solution calcination route, using Zn(NO3)2 as Zn source in the absence of any surfactants, templates or catalysts. This is the first example to prepare mushroom-like crystals as semiconductors, which are expected to show particular physical properties. The ZnO products were investigated by X-ray powder diffraction (XRD), scanning electron microscopy (SEM), Raman spectroscopy and photoluminescence (PL) spectrum measurements. A suitable concentration of Zn(NO3)2 solution was important for the growth of the mushroom-like products. The reported synthetic procedure is straightforward and inexpensive, and thus can be readily adopted to produce large quantities of mushroom-like ZnO microcrystals.

Porous MgO nanoplates have been successfully synthesized using a single-step Mg(NO 3 ) 2 solution calcination route without the assistance of any other additions. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) explorations indicated that the sizes of these nanoplates were ranging from 100 to 300 nm and the pores in them had diameters of about 2–50 nm. Nitrogen adsorption measurements for the prepared porous MgO nanoplates showed that the products had a BET (Brunauer–Emmett–Teller) surface area of 190 m 2 g −1 . PL (photoluminescence) spectrum of the products exhibited a visible light emission. Based on these investigations, the products were expected to find wide applications in biologic, catalytic and optoelectronic fields. Thermogravimetric (TG) analysis provided insight into the decomposition process of the Mg(NO 3 ) 2 solution precursor. A possible growth mechanism of the products was also explained.

A solution evaporation route has been successfully developed for the growth of copper nitrate hydroxide microcrystals using copper nitrate solution as the starting material in the absence of any surfactants or templates. The products were characterized by X-ray diffraction (XRD), infrared (IR) spectrum, scanning electron microscopy (SEM) and thermogravimetric (TG) analysis measurements. Controlled experiments suggested that the reaction temperature and solution concentration played an important role on the formation of the products. A possible formation mechanism of the products was also proposed.

Single-crystalline nanoplates of magnesium oxide were successfully synthesized through a calcinations route from the newly produced Mg(OH)2 precursors, which were directly prepared from the commercial bulk magnesium powders under a hydrothermal process in the absence of any additions. Scanning electron microscope (SEM) analysis indicated that the nanoplates were 2-6 m in average width and about 80 nm in thickness. Transmission electron microscopy (TEM) images revealed that there was large quantity of nanopores with diameters ranging from 5 to 40 nm distributed in these nanoplates. The room-temperature photoluminescence (PL) spectrum of the nanoplates illustrated a strong blue emission band at 416 nm and a weak green emission band at 559 nm. Brunauer-Emmett-Teller (BET) analysis exhibited a feature of high surface of 127.21 m2/g for the products. The fabrication mechanism of the product was also discussed.

Uniform bone-like MgO nanocrystals have been prepared via a solvothermal process using commercial Mg powders as the starting material in the absence of any catalyst or surfactant followed by a subsequent calcination. Field emission scanning electron microscopy (FE-SEM) and transmission electron microscopy (TEM) measurements indicate that the product consists of a large quantity of bone-like nanocrystals with lengths of 120–200 nm. The widths of these nanocrystals at both ends are in the range of 20–50 nm, which are 3–20 nm wider than those of the middle parts. Explorations of X-ray diffraction (XRD) and selected area electronic diffraction (SAED) exhibit that the product is high-quality cubic single-crystalline nanocrystals. The photoluminescence (PL) measurement suggests that the product has an intensive emission centered at 410 nm, showing that the product has potential application in optical devices. The advantages of our method lie in high yield, the easy availability of the starting materials and permitting large-scale production at low cost. The growth mechanism was proposed to be related with solvent’s oxidation in the precursor formation process and following nucleation and mass-transfer in the decomposition of the precursor.

The single-crystalline CdS dendrites have been fabricated from the reaction of CdCl2 and thiourea at 180 °C, in which glycine was employed as a soft template. The obtained products were explored by X-ray diffraction, scanning electron microscopy, transmission electron microscopy, and selected area electronic diffraction. The optical properties of CdS dendrites have been investigated by ultraviolet and visible light (UV-vis) and photoluminescence techniques. The investigations indicated that the dendrites were grown due to the anisotropic properties enhanced by the use of Glycine in the route.

Mammals have evolved neurophysiologic reflexes, such as coughing and scratching, to expel invading pathogens and noxious environmental stimuli. It is well established that these responses are also associated with chronic inflammatory diseases, including asthma and atopic dermatitis. However, the mechanisms by which inflammatory pathways promote sensations such as itch remain poorly understood. Here, we show that type 2 cytokines directly activate sensory neurons in both mice and humans. Further, we demonstrate that chronic itch is dependent on neuronal IL-4Rα and JAK1 signaling. We also observe that patients with recalcitrant chronic itch that failed other immunosuppressive therapies markedly improve when treated with JAK inhibitors. Thus, signaling mechanisms previously ascribed to the immune system may represent novel therapeutic targets within the nervous system. Collectively, this study reveals an evolutionarily conserved paradigm in which the sensory nervous system employs classical immune signaling pathways to influence mammalian behavior.

Mammals have evolved neurophysiologic reflexes such as scratching to expel invading pathogens and noxious environmental factors. It is also well established that these responses are associated with chronic inflammatory diseases such as atopic dermatitis. However, the mechanisms by which inflammatory pathways promote sensations such as itch remain poorly understood. Here, we show that the type 2 cytokines interleukin (IL)-4 and IL-13 directly stimulate sensory neurons and that chronic itch is dependent on neuronal IL-4Rα, the shared receptor subunit for IL-4 and IL-13. Based on our understanding of IL-4Rα signaling in immune cells, we hypothesized that downstream Janus kinase (JAK) signaling in sensory neurons would be a critical mediator of itch. Indeed, we find that both systemic JAK inhibition and interruption of JAK signaling in the nervous system can dramatically reduce itch. Further, sensory neuron-specific genetic deletion of JAK1 results in robust abatement of chronic itch in mice. Finally, in proof-of-concept translational studies, patients with recalcitrant chronic itch improved rapidly and dramatically in response to JAK inhibition. Thus, signaling mechanisms previously ascribed to the immune system may represent novel targets within the sensory nervous system for the treatment of pathologic sensory responses. Collectively, these studies reveal an evolutionarily conserved paradigm in which the sensory nervous system employs classical immune signaling pathways to influence mammalian behavior.

Peptide LSARLAF (LSA) can bind and activate integrin αIIbβ3 in the absence of 'inside-out' signal. The active αIIbβ3 mediates 'outside-in' signaling that elicits platelet aggregation, granule secretion and TxA2 production. Here we identify the membrane glycoproteins which mediate LSA-induced platelet activation other than αIIbβ3, and determine the roles of Src, PLCγ2, FcRγ-chain, and SLP-76 in LSA-induced platelet activation.Ligand-receptor binding assay was performed to study the effect of peptide LSA or its control peptide FRALASL (FRA) on integrins binding to their ligands. Spreading of CHO cells expressing αIIbβ3 or αVβ3 on immobilized fibrinogen was measured in the presence of LSA or FRA. Washed β3, Src, FcRγ-chain, LAT and SLP-76 deficient platelets aggregation and secretion were tested in response to LSA.Ligand-receptor binding assay indicated that LSA promoted the binding of multiple ligands to αIIbβ3 or αVβ3. LSA also enhanced CHO cells with αIIbβ3 or αVβ3 expression spreading on immobilized fibrinogen. β3 deficient platelets failed to aggregate and secrete in response to LSA. The phosphorylation of PLCγ2 and Syk was also β3 dependent. Src, FcRγ-chain, LAT and SLP-76 deficient platelets did not aggregate, secrete ATP or produce TxA2 in response to LSA.LSA-induced platelet activation is β3 dependent, and signaling molecules Src, FcRγ-chain, SLP-76 and LAT play crucial roles in LSA-induced β3 mediated signaling.

Integrin αIIbβ3 mediated bidirectional signaling plays a critical role in thrombosis and haemostasis. Signaling mediated by the β3 subunit has been extensively studied, but αIIb mediated signaling has not been characterized. Previously, we reported that platelet granule secretion and TxA2 production induced by αIIb mediated outside-in signaling is negatively regulated by the β3 cytoplasmic domain residues R(724)KEFAKFEEER(734). In this study, we identified part of the signaling pathway utilized by αIIb mediated outside-in signaling. Platelets from humans and gene deficient mice, and genetically modified CHO cells as well as a variety of kinase inhibitors were used for this work. We found that aggregation of TxA2 production and granule secretion by β3Δ724 human platelets initiated by αIIb mediated outside-in signaling was inhibited by the Src family kinase inhibitor PP2 and the PI3K inhibitor wortmannin, respectively, but not by the MAPK inhibitor U0126. Also, PP2 and wortmannin, and the palmitoylated β3 peptide R(724)KEFAKFEEER(734), each inhibited the phosphorylation of Akt residue Ser473 and prevented TxA2 production and storage granule secretion. Similarly, Akt phosphorylation in mouse platelets stimulated by the PAR4 agonist peptide AYPGKF was αIIbβ3-dependent, and blocked by PP2, wortmannin and the palmitoylated peptide p-RKEFAKFEEER. Akt was also phosphorylated in response to mAb D3 plus Fg treatment of CHO cells in suspension expressing αIIbβ3-Δ724 or αIIbβ3E(724)AERKFERKFE(734), but not in cells expressing wild type αIIbβ3. In summary, SFK(s) and PI3K/Akt signaling is utilized by αIIb-mediated outside-in signaling to activate platelets even in the absence of all but 8 membrane proximal residues of the β3 cytoplasmic domain. Our results provide new insight into the signaling pathway used by αIIb-mediated outside-in signaling in platelets.

Introduction: Retinoid receptors are nuclear hormone receptors which are dynamically regulated during terminal myeloid maturation. Retinoic acid receptor α (RARA) is the target of at least ten fusion proteins that lead to acute promyelocytic leukemia (APL). All trans-retinoic acid (ATRA) has been thought to be the principle natural ligand for RARs and it has been used for the treatment of patients with APL. However, the enzymatic pathways that regulate natural retinoids metabolism in hematopoietic cells have not been well defined. ATRA is synthesized from vitamin A through two sequential steps. Vitamin A is oxidized by an alcohol dehydrogenase to yield retinal, which is then irreversibly oxidized by an aldehyde dehydrogenase (ALDH) to generate retinoic acid (RA). At least 19 different human ALDHs have been identified. Among them, ALDH1A1, ALDH1A2 and ALDH1A3 have been shown to oxidize all trans-retinal to ATRA with high affinity, which can be inhibited by diethylaminobenzaldehyde (DEAB). Whether other ALDHs participate in RA metabolism is unknown. Our study identified two distinct retinoid metabolism pathways that are active in bone marrow (BM) progenitors and in macrophages (Mφ). Methods: Gal4-UAS reporter system was used to detect natural RARA ligands in mouse primary hematopoietic cells. We transduced UAS-GFP mouse BM cells with retrovirus that expresses a fusion protein containing the DNA binding domain of Gal4 (which recognizes the UAS promoter) and the ligand binding domain of RARA. An IRES mCherry cassette was included for normalization purposes. Transduced cells were cultured in vitro, or transplanted into lethally irradiated recipient mice. Cells with intracellular RARA ligands activate GFP expression. GFP and mCherry expression were evaluated by flow cytometry. Real-time PCR and Affymetrix array were used to quantify ALDH expression. We identified RARA ligands by mass spectrometry (MS). Results: In vitro, we found that both mouse BM Kit+ cells (Kit+ progenitors) and BM-derived macrophages (BMMφ) could synthesize active RARA ligands via different pathways, but only when the cell culture media was supplemented with vitamin A. Kit+ progenitors utilize DEAB-sensitive ALDH pathways, whereas BMMφ use DEAB-insensitive pathways. By real-time PCR we found Kit+ progenitors have high expression of Aldh1a1, Aldh1a2 and Aldh1a3, whereas BMMφ have no detectable expression of these enzymes. We compared gene expression in Kit+ progenitors and BMMφ by Affymetrix profiling and found that Aldh3b1 was overexpressed in BMMφ. Ectopic expression of Aldh3b1 in 293T cells resulted activation of the same GFP reporter, which could be abrogated by two different antagonist, Ro41-5253 and BMS493, suggesting that Aldh3b1 generated an RARA specific ligand, which we subsequently identified as ATRA via MS. Reciprocally, we found that siRNA knock down of Aldh3b1 in BMMφ reduced the transactivation of the RARA-dependent GFP reporter. The X-RARA fusions have been proposed to act via dominant-negative mechanisms, decreasing retinoid-dependent transcription and myeloid maturation. Surprisingly, in vivo, only rare GFPdim cells were observed in BM cells and no GFP positive cells in peritoneal Mφ of UAS-GFP/Gal4-RARA transplant mice. As positive control, we treated mice with ATRA and observed a dose-dependent GFP increase in both cell types, suggesting that the in vivo reporter can respond to ATRA, but ATRA is not synthesized during adult hematopoiesis, and that dominant-negative inhibition of ATRA-dependent transcription may not be the predominant pathogenic effect of the X-RARA fusion oncoproteins. Conclusion: We have found that at least two distinct enzymatic pathways may be utilized in primary hematopoietic cells to synthesize active RARA ligands from vitamin A. Mouse BM Kit+ progenitors predominantly employ a set of DEAB-sensitive enzymes (Aldh1a1, Aldh1a2 and Aldh1a3), whereas Mφ utilize DEAB-insensitive pathways. We identified Aldh3b1 as a likely candidate and shown that it is capable of ATRA synthesis in vitro. Although these enzymes are expressed in primary BM cells, we found that this does not result in active intracellular RARA ligands in monocytes or Mφ in vivo, suggesting that the rate-limiting step in retinoid synthesis in vivo is likely to involve additional enzymes required for intracellular transport of protein-bound, serum-available vitamin A. Disclosures No relevant conflicts of interest to declare.

The X-RARA fusion proteins have reduced sensitivity to all-trans-retinoic acid (ATRA), and have been proposed to act by decreasing retinoid-dependent transcription required for myeloid maturation.1, 2, 3 We therefore sought to determine whether maturing myeloid cells are exposed to natural retinoid ligands in vivo. Surprisingly, we detected a paucity of natural retinoids capable of transactivating RARA-dependent transcription in adult mouse bone marrow cells, and the trace activity we observed tended to be in erythroid-lineage cells, rather than in myeloid-progenitors. This suggests that the resistance to retinoid-mediated transactivation likely has a limited role in X-RARA-dependent leukemogenesis because natural retinoids are largely absent during normal myeloid maturation.

We sought to determine whether bexarotene can be combined with decitabine in elderly and relapsed AML patients. Both drugs have been shown to be well tolerated in acute myeloid leukemia (AML) patients as single agents, and these agents have non-overlapping mechanisms and side-effect profiles; bexarotene activates transcriptional effects of RXRA through hetero- and homodimers, while decitabine is thought to act through DNA hypomethylation. Furthermore, through Affymetrix expression array profiling of 111 AML patients and Nanostring analysis of 7 MDS and AML patients, we observed consistently elevated levels of RXRA relative to RARA, suggesting that a ligand specific for RXR may be more effective to induce AML differentiation than the RARA ligand ATRA. We treated 18 elderly (≥ 60 years old) or relapsed AML patients in 3+3 dose escalating bexarotene cohorts: 100 mg/m2/day, 200 mg/m2/day, 300 mg/m2/day. All patients were treated with decitabine 20 mg/m2IV on days 1-5 of 28 day cycles. All patients were monitored for hypertriglyceridemia and hypothyroidism, and treated accordingly. The average age was 73, the average performance status was 1, an adverse karyotype was observed in 9 patients, and 12 patients had relapsed after prior therapy. Only one patient experienced a dose limiting toxicity (grade 3 fatigue) and 8 patients were treated with the maximum dose (myelosuppression, infection, differentiation syndrome, hypertriglyceridemia, hyperlipidemia, hypothyroidism, nausea, weight loss and reversible electrolyte abnormalities were not considered dose limiting). The overall response rate was 22%: 1 patient achieved complete remission with incomplete count recovery (CRi) and 3 patients achieved blast reduction greater than 50% (partial response, PR). In addition, six patients achieved stable disease (SD). Patients with CRi, PR, or SD completed an average of 4.25 cycles, while other patients completed an average of 1.2 cycles. Of note, 3 patients successfully transitioned to allogeneic transplant following therapy (average age 68). We correlated ex vivo bexarotene sensitivity with clinical response. Bone marrow cells were collected on day 0 and day 3 of bexarotene therapy (during cycle 1, decitabine was administered on day 3 after bone marrow collection) and co-cultured with irradiated MS5 murine stromal cells for 72hrs with or without further bexarotene treatment. We used flow cytometry to compare CD11b expression in cells treated with and without bexarotene ex vivo, and compared expression between samples collected on day 0 vs day 3 (in vivo treatment). Bexarotene increased CD11b expression greater in the 4 responding patients vs non-responders (fold increase in CD11b: ex vivo average 2.1 ± 0.3 vs 1.1 ± 0.1 fold, p < 0.003; and in vivo 1.6 ± 0.3 vs 0.7 ± 0.2 fold, p < 0.03; increase in absolute percentage of CD11b+ cells: ex vivo average 24% ± 2.6% vs 0.7% ± 1%, p < 0.001; and in vivo 13.6% ± 4% vs -3.6% ± 2.2%, p < 0.002). Furthermore, all 4 responding patients demonstrated an equivalent or increased induction of CD11b when treated ex vivo with ATRA compared with bexarotene. These results show that bexarotene, a retinoid which selectively binds to and activates RXRs, but not RARs, can be safely combined with decitabine in relapsed and refractory AML patients. This combination leads to partial response in a subset of patients, is well tolerated, and can bridge elderly patients to allogeneic transplant. Because ex vivo bexarotene treatment identified all patients achieving a PR, further studies should focus on patients who display ex vivo sensitivity. Finally, the mechanism of RXRA-activated differentiation is likely to be through the RXRA/RARA heterodimer, as all 4 patients who responded to bexarotene also responded to ATRA when tested ex vivo. Disclosures: Welch: Eisai: Research Funding. Off Label Use: Bexarotene for the treatment of AML. Abboud:Ariad, Alexion, Novartis, Teva: Honoraria, Speakers Bureau. Stockerl-Goldstein:Celgene : Speakers Bureau; Millennium: Speakers Bureau.

Abstract 3252 PTEN (Phosphatase and tensin homologue deleted in chromosome 10) is a well-known tumor suppressor. PTEN dephosphorylates PtdIns(3,4,5)P3 into PtdIns(4,5)P2 and thereby negatively regulates PIP3-downstream signaling. According to present studies, PTEN is also a protein tyrosine phosphatase. In 2010 (Blood. 2010;116(14):2579–81), we reported that hematopoietic specific PTEN deficiency causes an increased platelet count, a shortened tail vein bleeding time, and enhanced platelet activation in response to stimulation by collagen and other agonists along with augmented collagen-induced phosphorylation of platelet Akt at Ser473. The PI3K specific inhibitor LY294002 almost completely blocked collagen-induced WT platelet aggregation, but had less effect on PTEN-deficient platelets. This result implies that PTEN may regulate collagen-induced platelet activation via both PI3K/Akt signaling and an yet to be identified pathway. In this study, we investigated the mechanism underlying PTEN regulation of collagen-induced platelet activation. It is well-known that the GPVI receptor signaling cascade is similar to that of T- and B-cell immune receptors. This signaling cascade relies on the formation of a signalosome composed of the transmembrane adapter LAT (linker for activation of T cells), the two cytosolic adapters SLP-76 and Gads, and the activation of PLCγ2. Accordingly, we investigated the phosphorylation of LAT, SLP-76 and PLCγ2 in PTEN-deficient and WT platelets in response to low level collagen (2μg/ml), with and without LY294002 pre-incubation. The platelet lysate was immunoprecipitated with 4G10, a specific anti-phosphotyrosine monoclonal antibody, to detect tyrosine phosphorylation. Interestingly, we found that in the presence of LY294002, the phosphorylation levels of LAT, SLP-76 and PLCγ2 were very high in PTEN-deficient platelets while the phosphorylation of these molecules was hardly detectable in WT platelets, all in response to low level collagen. These results suggested that PTEN may also regulate platelet activation through LAT, SLP-76 and PLCγ2. This hypothesis was supported by co-immunopricipitation experiments demonstrating that PTEN interacts with LAT in platelets upon collagen stimulation. Moreover, we also found that PTEN Ser380 phosphorylation was correlated with collagen-induced WT platelet activation without LY294002, and PTEN Ser380 phosphorylation could be inhibited by CK2 inhibitor DMAT and PDK1 inhibitor II, but not by Akt inhibitor SH6 or the GSK3β inhibitor LiCl. In contrast, the PDK1 inhibitor II blocked collagen-induced activation of CK2. Radioactivity assays indicated that collagen-induced enzyme activity activation of CK2 was blocked by the PDK1 inhibitor II. Ser380 phosphorylation is thought to reflect the loss of PTEN phosphatase function. So, because inhibition of PI3K, and PDK1 do not result in phosphorylation of PTEN S380 and the resulting loss of PTEN phosphatase function, it may be true that PDK1 inactivates PTEN. Therefore, we conclude that the mechanism of PTEN involvement in collagen-induced platelet activation is complicated, and PTEN plays a key role in GPVI mediated platelet activation probably through down-regulating both the PI3K/Akt and PI3K-PDK1-LAT pathways. We also investigated PTEN's function in αIIbβ3 mediated platelet activation. Platelet spreading and clot retraction experiments were performed. We found that PTEN deficiency significantly promoted clot retraction, but had no effect on platelet spreading on immobilized fibrinogen. Moreover, U46619 and thrombin induced PTEN ser380 phosphorylation was inhibited in β3 deficient platelet. These results indicate that PTEN is also involved in αIIbβ3 mediated outside-in signaling. Although the molecular mechanism of PTEN modulation of αIIbβ3 mediated outside-in signaling is unclear, our results may partially explain why PTEN deficiency also enhances platelet aggregation and secretion in response to agonists other than collagen. Disclosures: No relevant conflicts of interest to declare.

Abstract 4011 Poster Board III-947 Bidirectional signaling is an essential feature of integrin αIIbβ3 function. Outside-in signal transduction mediated by αIIbβ3 is required for normal hemostasis, clot retraction, platelet aggregation and spreading. In our previous work (Blood. 2005; 105: 4345-52), we showed that αIIb-mediated outside-in signaling is enhanced in human platelets (designated VTGβ3Δ724) lacking the terminal 39 residues of the β3 cytoplasmic tail. This enhanced signaling was detected as thromboxane A2 (TxA2) production and granule secretion, and required ligand cross-linking of αIIbβ3 and platelet aggregation. This αIIbβ3-mediated outside-in signaling was specifically inhibited by a palmitoylated, but not a scrambled or non-palmitoylated version of a peptide corresponding to β3 cytoplasmic domain (CD) residues R724-R734. Inhibition presumably occurred because the corresponding region within the β3 CD (RKEFAKFEEER) can bind to the CD of αIIb and negatively regulate αIIb-mediated outside-in signaling. In this study, we elucidate part of the signaling pathway(s) underlying these αIIb-elicited events. The data presented here demonstrate that D3+Fg-induced TxA2 production and granule secretion by VTGβ3Δ724 platelets are Src family kinase(s) (SFK), PI3K, PKC and intracellular Ca2+ flux, but not ERK dependent. Further biochemical analysis demonstrated that D3+Fg treatment of control platelets ± 10U/ml apyrase and 75mM indomethacin, induced phosphorylation of ERK and slight phosphorylation of p38, but not Akt. ERK activation was not dependent on a SFK(S) or PI3K since 10mM SFK of the inhibitor PP2 and 100nM PI3K inhibitor wortmannin did not block phosphorylation of ERK. However, Akt, and p38 were intensely phosphorylated in VTGβ3Δ724 platelets treated with D3+Fg ± apyrase and indomethacin, meaning that activation of Akt, and p38 appears to be directly coupled to αIIb-mediated signaling, and not coupled to TxA2 and ADP-driven signaling. Phosphorylation of p38 and Akt, but not ERK, was totally blocked by 10mM PP2. Wortmannin (100nM) inhibited phosphorylation of Akt, but not P38 and ERK. Therefore, SFK-PI3K/Akt-PKC play an important role in αIIb-mediated signaling. Akt phosphorylation in VTGβ3Δ724 platelets stimulated by D3+Fg was also specifically inhibited by a palmitoylated β3R724-R734 peptide but not by a palmitoylated scrambled control peptide, which further proves that SFK-PI3K signaling pathway is mediated specifically by αIIb. The role of αIIb-mediated SFK-dependent PI3K/Akt signaling was investigated in normal washed platelets by stimulating those platelets with a level of the PAR4 peptide that caused secretion-dependent aggregation. The aggregation initiated αIIbβ3-mediated outside-in signaling in both mouse and human washed platelets. The PAR4 peptide-induced Akt phosphorylation, TxA2 production and granule secretion were αIIbβ3, aggregation, and PI3K-dependent, in the absence of ADP and TxA2 initiated signaling. The palmitoylated peptide β3R724-R734 (10mM), but not a control peptide blocked ATP secretion, TxA2 production and Akt phosphorylation in normal platelets stimulated with the PAR4 peptide. Therefore, the αIIb-initiated SFK(s), PI3K/Akt, PKC signaling pathway is responsible for TxA2 production and granule secretion, in both VTGβ3Δ724 and normal platelets. SFK-dependent p38 activation is also triggered by αIIb-mediated signaling, but it is not Akt-dependent and the function of p38 in αIIb-mediated signaling events is unknown. Finally, ERK activation is αIIbβ3-dependent, but not αIIbβ3-mediated; therefore ERK activation probably is an effect of non-αIIb-mediated contact-dependent signaling. The work presented here provides a new understanding of αIIbb3- outside-in signaling. Disclosures: No relevant conflicts of interest to declare.

Atopic dermatitis (AD) is a widespread, chronic skin disease associated with aberrant allergic inflammation. Current treatments involve either broad or targeted immunosuppression strategies. However, enhancing the immune system to control disease remains untested. We demonstrate that patients with AD harbor a blood natural killer (NK) cell deficiency that both has diagnostic value and improves with therapy. Multidimensional protein and RNA profiling revealed subset-level changes associated with enhanced NK cell death. Murine NK cell deficiency was associated with enhanced type 2 inflammation in the skin, suggesting that NK cells play a critical immunoregulatory role in this context. On the basis of these findings, we used an NK cell-boosting interleukin-15 (IL-15) superagonist and observed marked improvement in AD-like disease in mice. These findings reveal a previously unrecognized application of IL-15 superagonism, currently in development for cancer immunotherapy, as an immunotherapeutic strategy for AD.