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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.

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 presence of natural retinoids in vivo could influence the efficacy of pharmacologic agonists and antagonists. We found that natural RXRA ligands, but not RARA ligands, were present in murine MLL-AF9-derived myelomonocytic leukemias in vivo and that the concurrent presence of receptors and ligands acted as tumor suppressors. Pharmacologic retinoid responses could be optimized by concurrent targeting RXR ligands (e.g. bexarotene) and RARA ligands (e.g. all-trans retinoic acid, ATRA), which induced either leukemic maturation or apoptosis depending on cell culture conditions. Co-repressor release from the RARA:RXRA heterodimer occurred with RARA activation, but not RXRA activation, providing an explanation for the combination synergy. Combination synergy could be replicated in additional, but not all, AML cell lines and primary samples, and was associated with improved survival in vivo, although tolerability of bexarotene administration in mice remained an issue. These data provide insight into the basal presence of natural retinoids in leukemias in vivo and a potential strategy for clinical retinoid combination regimens in leukemias beyond acute promyelocytic leukemia. This work was supported by NIH R01 HL128447 (to JSW), NIH P50 CA171963 (Project 1, to JSW and DRP) by the Siteman Investment Program (to JSW), and grants from the Spanish Ministerio de Ciencia e Innovacion (MCI) (SAF201571878-REDT-NurCaMeIn, RTI2018-095928-B100) (to MR). The CNIC is supported by the MCI and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence (SEV2015-0505). Sí

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

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.

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.

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.

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.

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.