Pathway Agonists Research Articles

GLI1 Blockade Potentiates the Antitumor Activity of PI3K Antagonists in Lung Squamous Cell Carcinoma.

Lung squamous cell carcinoma (SCC), strongly associated with smoking, is treated primarily with traditional cytotoxic chemotherapy due to a lack of FDA-approved targeted agents available. Here, we identify the Hedgehog pathway transcription factor GLI1 as a critical driver of lung SCC. Analysis of human lung cancer datasets showed that GLI1 mRNA was highly expressed in human lung SCC and portended a poor prognosis. Inhibition of GLI1 in human lung SCC cell lines suppressed tumor cell clonogenicity and proliferation in culture and in vivo Addition of SHH ligand, SMO antagonists, or other Hedgehog pathway agonists did not affect GLI1 expression in lung SCC cells. However, GLI1 expression was modulated by either inhibition or activation of the PI3K and MAPK pathways. Furthermore, in vivo growth of SCC harboring amplifications of the PI3K gene PIK3CA was attenuated by antagonizing GLI1 and PI3K. Thus, a combinatorial therapeutic strategy that targets the PI3K-mTOR pathway and GLI1 may lead to effective outcomes for PI3K pathway-dependent cancers, in contrast to recent results of human trials with single-agent PI3K antagonists. Cancer Res; 77(16); 4448-59. ©2017 AACR.

Macrophage Migration Inhibitory Factor Limits Renal Inflammation and Fibrosis by Counteracting Tubular Cell Cycle Arrest.

Renal fibrosis is a common underlying process of progressive kidney diseases. We investigated the role of macrophage migration inhibitory factor (MIF), a pleiotropic proinflammatory cytokine, in this process. In mice subjected to unilateral ureteral obstruction, genetic deletion or pharmacologic inhibition of MIF aggravated fibrosis and inflammation, whereas treatment with recombinant MIF was beneficial, even in established fibrosis. In two other models of progressive kidney disease, global Mif deletion or MIF inhibition also worsened fibrosis and inflammation and associated with worse kidney function. Renal MIF expression was reduced in tubular cells in fibrotic compared with healthy murine and human kidneys. Bone marrow chimeras showed that Mif expression in bone marrow-derived cells did not affect fibrosis and inflammation after UUO. However, Mif gene deletion restricted to renal tubular epithelial cells aggravated these effects. In LPS-stimulated tubular cell cultures, Mif deletion led to enhanced G2/M cell-cycle arrest and increased expression of the CDK inhibitor 1B (p27Kip1) and of proinflammatory and profibrotic mediators. Furthermore, MIF inhibition reduced tubular cell proliferation in vitro In all three in vivo models, global Mif deletion or MIF inhibition caused similar effects and attenuated the expression of cyclin B1 in tubular cells. Mif deletion also resulted in reduced tubular cell apoptosis after UUO. Recombinant MIF exerted opposing effects on tubular cells in vitro and in vivo Our data identify renal tubular MIF as an endogenous renoprotective factor in progressive kidney diseases, raising the possibility of pharmacologic intervention with MIF pathway agonists, which are in advanced preclinical development.

Single-Center Experience Using Selexipag in a Pediatric Population.

Pulmonary arterial hypertension (PAH) is a rare and progressive disorder. Current treatment in the pediatric population includes phosphodiesterase 5 inhibitors (PDE-5i), endothelin receptor antagonists (ERA), and both inhaled and intravenous prostacyclin pathway agonists. As of December 22, 2015 the first oral prostacyclin pathway agonist, selexipag (Uptravi®), was FDA approved in the US. In this case series, we discuss our single-center experience using selexipag in a pediatric population, composed of both patients with idiopathic PAH, and patients with congenital heart disease and PAH.

Mutual reinforcement between telomere capping and canonical Wnt signalling in the intestinal stem cell niche

Critical telomere shortening (for example, secondary to partial telomerase deficiency in the rare disease dyskeratosis congenita) causes tissue pathology, but underlying mechanisms are not fully understood. Mice lacking telomerase (for example, mTR−/− telomerase RNA template mutants) provide a model for investigating pathogenesis. In such mice, after several generations of telomerase deficiency telomeres shorten to the point of uncapping, causing defects most pronounced in high-turnover tissues including intestinal epithelium. Here we show that late-generation mTR−/− mutants experience marked downregulation of Wnt pathway genes in intestinal crypt epithelia, including crypt base columnar stem cells and Paneth cells, and in underlying stroma. The importance of these changes was revealed by rescue of crypt apoptosis and Wnt pathway gene expression upon treatment with Wnt pathway agonists. Rescue was associated with reduced telomere-dysfunction-induced foci and anaphase bridges, indicating improved telomere capping. Thus a mutually reinforcing feedback loop exists between telomere capping and Wnt signalling, and telomere capping can be impacted by extracellular cues in a fashion independent of telomerase.

RSPOs facilitated HSC activation and promoted hepatic fibrogenesis

Roof plate-specific spondin (RSPO) proteins are potent Wnt pathway agonists and involve in a broad range of developmental and physiological processes. This study investigated the activities and mechanisms of RSPOs in liver fibrogenesis, especially in hepatic stellate cell (HSC) activation. HSC activation was assessed by fibrosis biomarker (α-smooth muscle actin and Collagen-I), phenotypic change (accumulation of lipid droplets), and increased proliferation. Similarly, Wnt pathway activity was evaluated by the expression of nuclear β-catenin and T cell-specific transcription factors (TCF) activity. We found RSPOs were overexpressed in human fibrotic liver tissue and the expressions were correlated with liver fibrosis stages. In vitro studies showed RSPOs level increased during HSC activation, and stimuli with RSPOs enhanced Wnt pathway activity and promoted HSC activation subsequently. Furthermore, in vivo experiments demonstrated that the knockdown of RSPOs suppressed both Wnt pathway activity and HSC activation. Interestingly, the inhibitor of the Wnt signaling pathway Dickkopf1 impairs RSPOs effects on HSCs. Taken together, our results revealed that RSPOs facilitated HSC activation and promote liver fibrogenesis by enhancing the Wnt pathway.

Abstract 38: Engineered Anisotropic Scaffolds Promote the Function of Cocultured Cardiomyocytes Derived From Human Pluripotent Stem Cells

Since the heart is effectively an anisotropic organ in which the cardiomyocytes (CM) are locally aligned in series, it is important to engineer cardiac tissues that promote CM alignment in order to closely mimic the architecture of the native tissue, as well as better mimic the cellular composition of the heart. The objective of this study was to define the role of anisotropic extracellular matrix cues on the organization and survival of human induced pluripotent stem cell-derived CMs (hiPSC-CMs) by co-culturing hiPSC-CMs and primary endothelial cells (ECs) on parallel-aligned microfibrillar scaffolds. The hiPSC-CMs were generated from hiPSCs using small molecule Wnt pathway agonists and antagonists. Subsequently, the hiPSC-CMs were sequentially seeded on day 15 after EC attachment. We cultured monocultures and cocultures on electrospun three-dimensional (3D) scaffolds of polycaprolactone (PCL) and polyethylene oxide (PEO) polymer blends with an average fiber diameter of 14 μm. Aligned scaffolds were fabricated by stretching the randomly oriented scaffolds by 300% of the original scaffold length. Randomly oriented fibrillar scaffolds had an average pore diameter of 17 μm when compared to the 36 μm pore diameter of aligned scaffolds. Our results indicate that alignment of co-cultured cells at a 5:1 hiPSC-CMs : EC ratio was promoted by anistropic 3D electrospun scaffolds when compared to similar random 3D electrospun scaffolds. Additionally, cocultured cells on aligned fibrillar scaffolds had a mean angle of orientation of 30.8°, relative to the direction of fibrils, which was similar to that of hiPSC-CM monocultures on aligned scaffolds (32.8°). In contrast, the degree of alignment of hiPSC-CMs on randomly oriented fibrillary scaffolds was 43.4°, which suggests a non-oriented population of cells. Aligned scaffolds also produced more synchronized cardiomyocyte contraction than random scaffold orientations, although both induced spontaneous contraction frequency of ~1Hz. This study highlights the importance of nanotopographical cues and intercellular interactions in mediating the morphology and contractility of hiPSC-CMs for treatment of cardiovascular diseases such as myocardial infarction.

Cover Picture: Synthesis and Evaluation of Osteogenic Oxysterols as Hedgehog Pathway Activators (ChemMedChem 7/2016)

The front cover picture shows how the stereochemical orientation of the C-21 methyl group in the oxysterol (OHC) scaffold can determine whether the OHC serves as an agonist of the hedgehog (Hh) signaling pathway (hedgehog image by Marina Maslennikova, iStockphoto). The natural configuration of the C-21 methyl is R, which is shown here in the structure of natural 23(S)-hydroxycholesterol (4, right), a potent, synthetic agonist of Hh signaling with the ability to promote osteogenic differentiation. Inversion of the C-21 methyl to the S configuration (16, left) completely abolished its ability to activate Hh signaling. These results provide essential structural insight for the future development of small molecule OHCs as Hh pathway agonists. More information can be found in the Full Paper by M. Kyle Hadden et al. on page 679 in Issue 7, 2016 (DOI: 10.1002/cmdc.201500550).

Assessment of neonatal platelet adhesion, activation, and aggregation

Acquired and inherited bleeding disorders may present in the neonatal period with devastating lifelong effects. Diagnosing bleeding disorders in the neonatal population could aid in preventing and treating the associated complications. However, currently available platelet function testing is limited in neonates, owing to difficulties in obtaining an adequate blood volume, a lack of normal reference ranges, and an incomplete understanding of the neonatal platelet functional phenotype. To develop small-volume, whole blood platelet function assays in order to quantify and compare neonatal and adult platelet function. Peripheral blood was obtained from healthy, full-term neonates at 24 h of life. Platelet activation, secretion and aggregation were measured via flow cytometry. Platelet adhesion and aggregation were assessed under static and flow conditions. As compared with adult platelets, peripheral neonatal platelet P-selectin expression and integrin glycoprotein IIbIIIa activation were significantly reduced in response to the G-protein-coupled receptor (GPCR) agonists thrombin receptor activator peptide-6 (TRAP-6), ADP, and U46619, and the immunoreceptor tyrosine-based activation motif (ITAM) signaling pathway agonists collagen-related peptide (CRP) and rhodocytin. Neonatal platelet aggregation was markedly reduced in response to TRAP-6, ADP, U46619, CRP and rhodocytin as compared with adult platelets. The extents of neonatal and adult platelet adhesion and aggregate formation under static and shear conditions on collagen and von Willebrand factor were similar. As compared with adult platelets, we found that neonatal platelet activation and secretion were blunted in response to GPCR or ITAM agonists, whereas the extent of neonatal platelet adhesion and aggregate formation was similar to that of adult platelets.

Induction of Human iPSC-Derived Cardiomyocyte Proliferation Revealed by Combinatorial Screening in High Density Microbioreactor Arrays.

Inducing cardiomyocyte proliferation in post-mitotic adult heart tissue is attracting significant attention as a therapeutic strategy to regenerate the heart after injury. Model animal screens have identified several candidate signalling pathways, however, it remains unclear as to what extent these pathways can be exploited, either individually or in combination, in the human system. The advent of human cardiac cells from directed differentiation of human pluripotent stem cells (hPSCs) now provides the ability to interrogate human cardiac biology in vitro, but it remains difficult with existing culture formats to simply and rapidly elucidate signalling pathway penetrance and interplay. To facilitate high-throughput combinatorial screening of candidate biologicals or factors driving relevant molecular pathways, we developed a high-density microbioreactor array (HDMA) – a microfluidic cell culture array containing 8100 culture chambers. We used HDMAs to combinatorially screen Wnt, Hedgehog, IGF and FGF pathway agonists. The Wnt activator CHIR99021 was identified as the most potent molecular inducer of human cardiomyocyte proliferation, inducing cell cycle activity marked by Ki67, and an increase in cardiomyocyte numbers compared to controls. The combination of human cardiomyocytes with the HDMA provides a versatile and rapid tool for stratifying combinations of factors for heart regeneration.

Design, Synthesis, and Structural Optimization of Lycorine-Derived Phenanthridine Derivatives as Wnt/β-Catenin Signaling Pathway Agonists.

Lycorine is a benzylphenethylamine-type alkaloid member of the Amaryllidaceae family. A lycorine derivative, HLY78, was previously identified as a new Wnt/β-catenin signaling pathway agonist that targets the DAX domain of axin. Herein, the structural optimization of HLY78 and analyses of the structure-activity relationships of lycorine-derived phenanthridine derivatives as agonists of the Wnt/β-catenin signaling pathway are presented. This research suggests that triazole groups are important pharmacophores for Wnt activation; triazole groups at C-8 and C-9 of phenanthridine compounds markedly enhanced Wnt activation. A C-11-C-12 single bond is also important for Wnt activation. On the basis of these findings, two Wnt agonists were designed and synthesized. The results for these agonists indicated that the combination of a 4-ethyldihydrophenanthridine skeleton and a triazole substituent improves Wnt activation. These compounds may be useful in further pharmacological or biological studies.

PTPS-07COMPETING MOLECULAR GENETIC FORCES: TRISOMY 21(DOWN SYNDROME, DS) AND PTCH1 MUTATION (GORLIN SYNDROME) IN A 21 MONTH-OLD WITH SHH DESMOPLASTIC/NODULAR MEDULLOBLASTOMA

BACKGROUND: Individuals with Trisomy 21 have a striking cancer distribution, with a 10- to 20- fold increased risk of acute leukemia compared to a markedly decreased incidence of solid tumors. Medulloblastoma, the most common malignant brain tumor of childhood, is particularly rare in the DS population, with only one published case. As demonstrated in a mouse model of DS, the supernumerary chromosome 21 is associated with cerebellar hypoplasia and a decreased number of external granule cell layer progenitor cells (EGLs). Treatment of these mice with sonic hedgehog (Shh) signaling pathway agonists promotes normalization of EGLs and improved cognitive functioning. PATIENT: We describe a 21 month-old male with DS with desmoplastic/nodular medulloblastoma (DNMB) - a tumor derived from Shh dysregulation and over-activation of EGLs. Immunohistochemistry revealed patchy cytoplasmic GAB-1 staining and patchy strong YAP-1 nuclear and cytoplasmic staining, further placing this DNMB into the new consensus molecular classification Shh subgroup. Additional testing revealed a de novo heterozygous germ line mutation in the PTCH1 gene encoding a tumor suppressor protein in the Shh pathway; both parents tested negative for this mutation. CONCLUSIONS: The reduced presence of EGLs in DS patients offers a plausible explanation for the disparity of medulloblastoma in this population. Conversely, patients with PTCH1 germ line mutations experience Shh pathway overstimulation resulting in Gorlin (Nevoid Basal Cell Carcinoma) Syndrome and an increased incidence of malignant transformation of EGLs leading to medulloblastoma formation. This represents the first documented report of an individual with DS (developmental failure of EGLs, decreased incidence of medulloblastoma) simultaneously carrying PTCH1 germ line mutation (overstimulated EGLs, increased incidence of medulloblastoma). Thus, we observe a highly unusual circumstance in which the PTCH1 mutation appears to ‘trump’ the effects of trisomy 21 in causation of Shh-activated medulloblastoma. Additional genomic studies are ongoing and will be presented.

Synthesis of 4-Ethyl-5-Methyl-5,6-Dihydrophenanthridine-8,9-Diol as the Key Intermediate of Potent Agonists of the Wnt Signalling Pathway

The synthesis of 4-ethyl-5-methyl-5,6-dihydro-phenanthridine-8,9-diol as the key intermediate of a series of potent Wnt signalling pathway agonists is reported. This synthesis features a consecutive aryl-aryl and N-aryl coupling, leading to 4-ethyl-5-methyl-8,9-dimethoxyphenanthridin-6-(5H)-one, whose structure was determined by spectroscopic analysis and X-ray crystallography, in a one-pot sequence with the presence of palladium catalyst and TFP (tri(2-furyl)phosphine) as the ligand.

414 Reversal of Opioid-Induced Constipation by Lubiprostone (Amitiza®) in Guinea Pig Ileum

Background & Aims: Brain-derived neurotrophic factor (BDNF) may play a critical role in gut motility. We aimed to investigate BDNF's physiologic effects on gut motility in slowtransit constipation (STC) and to explore the underlying molecular mechanisms. Methods: BDNF expression and alterations of colonic nerve fiber density in STC patients were first investigated. BDNF's effects on gastrointestinal motility of both BDNF+/− mice and loperamide-induced constipation mice were then examined in vivo and in vitro. Smooth muscle α-actin (α-SMA) expression, and nerve fiber, neuromuscular junction (NMJ), and smooth muscle cell (SMC) alterations were investigated. Finally, the effects of BDNF-induced TrkBphospholipase C/inositol trisphosphate (TrkB-PLC/IP3) pathway activation on gut motility were investigated. Results: In STC patients, BDNF expression and nerve fiber density were decreased, and mucosal nerve fiber ultrastructural degenerations were demonstrated. Gut motility was decreased in vivo and in vitro in BDNF+/− and constipation mice, with BDNF dose-dependently increasing gut motility. In BDNF+/− mice, α-SMA expression and nerve fiber density were decreased, and nerve fiber, NMJ, and SMC ultrastructural degenerations were observed. Finally, TrkB-PLC/IP3 pathway agonists dramatically attenuated BDNF's excitatory effect on gut motility, and exogenous BDNF induced an obvious increase in IP3 expression. Conclusions: BDNF plays an important regulatory role in gut motility in STC. It was mediated by altering the intestinal innervation structure, as well as smooth muscle secondary degeneration through a mechanism involving TrkB-PLC/IP3 pathway activation.

413 Brain-Derived Neurotrophic Factor Accelerates Gut Motility in Slow-Transit Constipation

Background & Aims: Brain-derived neurotrophic factor (BDNF) may play a critical role in gut motility. We aimed to investigate BDNF's physiologic effects on gut motility in slowtransit constipation (STC) and to explore the underlying molecular mechanisms. Methods: BDNF expression and alterations of colonic nerve fiber density in STC patients were first investigated. BDNF's effects on gastrointestinal motility of both BDNF+/− mice and loperamide-induced constipation mice were then examined in vivo and in vitro. Smooth muscle α-actin (α-SMA) expression, and nerve fiber, neuromuscular junction (NMJ), and smooth muscle cell (SMC) alterations were investigated. Finally, the effects of BDNF-induced TrkBphospholipase C/inositol trisphosphate (TrkB-PLC/IP3) pathway activation on gut motility were investigated. Results: In STC patients, BDNF expression and nerve fiber density were decreased, and mucosal nerve fiber ultrastructural degenerations were demonstrated. Gut motility was decreased in vivo and in vitro in BDNF+/− and constipation mice, with BDNF dose-dependently increasing gut motility. In BDNF+/− mice, α-SMA expression and nerve fiber density were decreased, and nerve fiber, NMJ, and SMC ultrastructural degenerations were observed. Finally, TrkB-PLC/IP3 pathway agonists dramatically attenuated BDNF's excitatory effect on gut motility, and exogenous BDNF induced an obvious increase in IP3 expression. Conclusions: BDNF plays an important regulatory role in gut motility in STC. It was mediated by altering the intestinal innervation structure, as well as smooth muscle secondary degeneration through a mechanism involving TrkB-PLC/IP3 pathway activation.

Hedgehog (Hh) Reporter Activity Assay

This protocol is for testing responses of a candidate cell line/cell lines to Hh ligands or Hh pathway agonists stimulation. This protocol can also be adapted to screen small molecule libraries or biologics that contain activities to either increase or decrease Hh pathway responses. Canonical Hh signaling activity transcriptionally induces Hh target genes that contain consensus Gli binding element. Hh-responsive cells transiently or stably expressing luciferase protein under the regulation of the Gli promoter element can be used to report stimulus-dependent Hh-pathway activity., 该方案用于测试候选细胞系/细胞系对Hh配体或Hh通路激动剂刺激的反应。 该方案还可以适于筛选含有增加或降低Hh通路反应的活性的小分子文库或生物制剂。 规范Hh信号活动转录诱导含有共有Gli结合元件的Hh靶基因。 在Gli启动子元件的调节下瞬时或稳定表达荧光素酶蛋白的Hh反应性细胞可用于报告刺激依赖性Hh-途径活性。

Wnt Reporter Activity Assay

This protocol is for testing responses of a candidate cell line/cell lines to Wnt ligands or Wnt pathway agonists stimulation. This protocol can also be adapted to screen small molecule libraries or biologics that contain activities to either increase or decrease Wnt pathway responses. Canonical Wnt signaling activity transcriptionally induces Wnt target genes that contain concensus TCF/LEF binding element. Wnt pathway activity responsive cells transiently or stably expressing luciferase proteins under the TCF/LEF promoter element can be used to report stimulus-dependent Wnt-pathway activity. We acquired the TopFlash (TCL/LEF-Firefly luciferase) construct from Addgene.

Hedgehog Pathway Agonism: Therapeutic Potential and Small‐Molecule Development

The Hedgehog (Hh) pathway is a developmental signaling pathway that plays multiple roles during embryonic development and in adult tissues. Constitutive Hh signaling has been linked to the development and progression of several forms of cancer, and the application of small-molecule pathway inhibitors as anticancer chemotherapeutics is well studied and clearly defined. Activation of the Hh pathway as a therapeutic strategy for a variety of degenerative or ischemic disorders has also been proposed; however, the development of small-molecule Hh agonists has received less attention. The goal of this review is to highlight the recent evidence supporting the therapeutic potential of Hh pathway activators and to provide a comprehensive overview of small-molecule pathway agonists.

The JAK2/STAT3 and mitochondrial pathways are essential for quercetin nanoliposome-induced C6 glioma cell death.

The formulation of quercetin nanoliposomes (QUE-NLs) has been shown to enhance QUE antitumor activity in C6 glioma cells. At high concentrations, QUE-NLs induce necrotic cell death. In this study, we probed the molecular mechanisms of QUE-NL-induced C6 glioma cell death and examined whether QUE-NL-induced programmed cell death involved Bcl-2 family and mitochondrial pathway through STAT3 signal transduction pathway. Downregulation of Bcl-2 and the overexpression of Bax by QUE-NL supported the involvement of Bcl-2 family proteins upstream of C6 glioma cell death. In addition, the activation of JAK2 and STAT3 were altered following exposure to QUE-NLs in C6 glioma cells, suggesting that QUE-NLs downregulated Bcl-2 mRNAs expression and enhanced the expression of mitochondrial mRNAs through STAT3-mediated signaling pathways either via direct or indirect mechanisms. There are several components such as ROS, mitochondrial, and Bcl-2 family shared by the necrotic and apoptotic pathways. Our studies indicate that the signaling cross point of the mitochondrial pathway and the JAK2/STAT3 signaling pathway in C6 glioma cell death is modulated by QUE-NLs. In conclusion, regulation of JAK2/STAT3 and ROS-mediated mitochondrial pathway agonists alone or in combination with treatment by QUE-NLs could be a more effective method of treating chemical-resistant glioma.

Drugging Hedgehog: signaling the pathway to translation

First discovered in Drosophila, the Hedgehog signaling pathway controls a wide range of developmental processes and is implicated in a variety of cancers. The success of a screen for chemical modulators of this pathway, published in 2002, opened a new chapter in the quest to translate the results of basic developmental biology research into therapeutic applications. Small molecule pathway agonists are now used to program stem cells, whilst antagonists are proving effective as anti-cancer therapies.

The miR-99∼125 Polycistrons Promote Leukemogenesis in a Cell-Context Dependent Manner by Shifting the Balance Between TGFβ- and Wnt-Signaling

Abstract 109Three human miR-99∼125 clusters, each containing one miR-99/100, let-7 and miR-125 family member in identical polycistronic configuration, are located within or next to long non-coding RNA (lncRNA) host genes on distinct chromosomal regions. In this study, we investigated the integrative function of these three polycistron-miRNAs. Expression profiling in sorted leukemic blasts indicated an enrichment of mature miR-99∼125 miRNAs in acute megakaryoblastic leukemia (AMKL) and promyelocytic leukemia (APL). We demonstrated that intronic miR-99∼125 miRNAs are produced from one primary transcript with their lncRNA host genes and that the leukemogenic transcription factor and stem cell regulator HOXA10 acts as an upstream transcriptional activator. Therefore, we assessed the consequences of concerted activation of miR-125b, miR-99/100 and let-7 in the megakaryocytic and myelomonocytic system.During megakaryocytic in vitro differentiation of hematopoietic stem and progenitor cells (HSPCs), lentiviral overexpression of the tricistron (miR-99a/let-7c/miR-125b-2) or miR-125b-2 alone increased proliferation and survival of megakaryocytic cells. Megakaryocytic/erythroid progenitors (MEP) were expanded upon tricistron and miR-125b-2 overexpression, and not by miR-99a and/or let-7c overexpression. In an unbiased triple-transduction RGB-competition assay using distinct color-marked (red/green/blue) monocistronic miRNA constructs, miR-125b-2 alone or in combination with miR-99a or let-7 conferred a selective growth advantage to murine and human HSPCs. In murine transplantation experiments, only combined expression of all three cluster-miRNAs led to the expansion and retention of an immature Gr-1(low)/Mac-1(+)/B220(-) cell population in the bone marrow. Similarly when the miR-99a/let-7c/miR-125b-2 tricistron-transduced HSPCs differentiated along the myelomonocytic lineage (liquid culture and methylcellulose colony-forming assay), we observed a major cell population of monomorphic, non-adherent cells devoid of granulocytic and monocytic markers, which was not present in the miR-99a-, let-7c- or miR-125b-2 single transduced cells.Global gene expression profiling followed by qRT-PCR-validation of transduced HSPCs identified high confidence targets commonly or individually downregulated by the three miRNAs. Among these targets, we found TGFβ pathway agonists and Wnt antagonists to be enriched. miR-125b and -even stronger- the tricistron decreased the reporter activity of a SMAD response element in 293T cells treated with TGFβ and increased the activity of a TCF response element in synergy with a Wnt-activator (GSK3-inibitor). Western blotting and luciferase reporter assays demonstrated cooperative targeting of TGFBR1, ALK7, BMPR1a, BMPR2, SMAD2, SMAD4 and APC by the tricistron. Strikingly, inhibition of miR-125b by a lentiviral decoy sensitized TGFβ-resistant AMKL-cell lines (CMK and CMY) to TGFβ-mediated apoptosis and proliferation arrest. Conversely, overexpression of miR-125b or the tricistron rendered MV4:11 and NB4 cells resistance to TGFβ. miR-125b or the tricistron-transduced HSPCs displayed a marked increase in cell viability and cells in S-Phase upon TGFβ-treatment during megakaryocytic differentiation. The application of a TGFβ-inhibitor phenocopied the effects of miR-125b or the tricistron during megakaryocytic differentiation. These effects were not further enhanced by combined Wnt activation. In contrast, recapitulating the effects of the polycistron in the myelomonocytic lineage required Wnt-activation.Collectively we deciphered the functional linkage of miR-99/100, let-7 and miR-125, which are produced from one primary transcript with their lncRNA host genes and are regulated by the stem cell regulator HOXA10. Our study creates a microRNA interaction network, wherein the concerted action of the three miRNAs converges at the TGFβ pathway by a combinatorial block of this pathway. During myelomonocytic commitment only the combination of all polycistron-miRNAs enhanced Wnt signaling to inhibit differentiation. These synthetic phenotypes form an epistatic circuit regulating stemness and developmental fate. Disclosures:No relevant conflicts of interest to declare.