scholarly journals Modulation of the Cardiac Sodium Channel NaV1.5 Peak and Late Currents by NAD+ Precursors

2020 ◽  
Author(s):  
Daniel S. Matasic ◽  
Jin-Young Yoon ◽  
Jared M. McLendon ◽  
Haider Mehdi ◽  
Mark S. Schmidt ◽  
...  
Keyword(s):  
Sodium Channel ◽  
Membrane Trafficking ◽  
Cardiac Electrophysiology ◽  
Phosphorylation Site ◽  
Sirtuin 1 ◽  
Hek293 Cells ◽  
Mutant Form ◽  
Wild Type ◽  
Cardiac Sodium Channel

ABSTRACTRationaleThe cardiac sodium channel NaV1.5, encoded by SCN5A, produces the rapidly inactivating depolarizing current INa that is responsible for the initiation and propagation of the cardiac action potential. Acquired and inherited dysfunction of NaV1.5 results in either decreased peak INa or increased residual late INa (INa,L), leading to tachy/bradyarrhythmias and sudden cardiac death. Previous studies have shown that increased cellular NAD+ and NAD+/NADH ratio increase INa through suppression of mitochondrial reactive oxygen species and PKC-mediated NaV1.5 phosphorylation. In addition, NAD+-dependent deacetylation of NaV1.5 at K1479 by Sirtuin 1 increases NaV1.5 membrane trafficking and INa. The role of NAD+ precursors in modulating INa remains unknown.ObjectiveTo determine whether and by which mechanisms the NAD+ precursors nicotinamide riboside (NR) and nicotinamide (NAM) affect peak INa and INa,Lin vitro and cardiac electrophysiology in vivo.Methods and ResultsThe effects of NAD+ precursors on the NAD+ metabolome and electrophysiology were studied using HEK293 cells expressing wild-type and mutant NaV1.5, rat neonatal cardiomyocytes (RNCMs), and mice. NR increased INa in HEK293 cells expressing NaV1.5 (500 μM: 51 ± 18%, p=0.02, 5 mM: 59 ± 22%, p=0.03) and RNCMs (500 µM: 60 ± 26%, p=0.02, 5 mM: 75 ± 39%, p=0.03) while reducing INa,L at the higher concentration (RNCMs, 5 mM: −45 ± 11%, p=0.04). NR (5 mM) decreased NaV1.5 K1479 acetylation but increased INa in HEK293 cells expressing a mutant form of NaV1.5 with disruption of the acetylation site (NaV1.5-K1479A). Disruption of the PKC phosphorylation site abolished the effect of NR on INa. Furthermore, NAM (5 mM) had no effect on INa in RNCMs or in HEK293 cells expressing wild-type NaV1.5, but increased INa in HEK293 cells expressing NaV1.5-K1479A. Dietary supplementation with NR for 10-12 weeks decreased QTc in C57BL/6J mice (0.35% NR: −4.9 ± 2.0%, p=0.26; 1.0% NR: −9.5 ± 2.8%, p=0.01).ConclusionsNAD+ precursors differentially regulate NaV1.5 via multiple mechanisms. NR increases INa, decreases INa,L, and warrants further investigation as a potential therapy for arrhythmic disorders caused by NaV1.5 deficiency and/or dysfunction.

scholarly journals The N-terminal domain of the human eIF2β subunit and the CK2 phosphorylation sites are required for its function

2006 ◽  
Vol 394 (1) ◽  
pp. 227-236 ◽  
Author(s):  
Franc Llorens ◽  
Anna Duarri ◽  
Eduard Sarró ◽  
Nerea Roher ◽  
Maria Plana ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Hela Cells ◽  
Mutant Cell ◽  
Phosphorylation Site ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Mutant Form ◽  
Wild Type ◽  
Main Site

CK2 (protein kinase CK2) is known to phosphorylate eIF2 (eukaryotic translation initiation factor 2) in vitro; however, its implication in this process in living cells has remained to be confirmed. The combined use of chemical inhibitors (emodin and apigenin) of CK2 together with transfection experiments with the wild-type of the K68A kinase-dead mutant form of CK2α evidenced the direct involvement of this protein kinase in eIF2β phosphorylation in cultured HeLa cells. Transfection of HeLa cells with human wild-type eIF2β or its phosphorylation site mutants showed Ser2 as the main site for constitutive eIF2β phosphorylation, whereas phosphorylation at Ser67 seems more restricted. In vitro phosphorylation of eIF2β also pointed to Ser2 as a preferred site for CK2 phosphorylation. Overexpression of the eIF2β S2/67A mutant slowed down the rate of protein synthesis stimulated by serum, although less markedly than the overexpression of the Δ2–138 N-terminal-truncated form of eIF2β (eIF2β-CT). Mutation at Ser2 and Ser67 did not affect eIF2β integrating into the eIF2 trimer or being able to complex with eIF5 and CK2α. The eIF2β-CT form was also incorporated into the eIF2 trimer but did not bind to eIF5. Overexpression of eIF2β slightly decreased HeLa cell viability, an effect that was more evident when overexpressing the eIF2β S2/67A mutant. Cell death was particularly marked when overexpressing the eIF2β-CT form, being detectable at doses where eIF2β and eIF2β S2/67A were ineffective. These results suggest that Ser2 and Ser67 contribute to the important role of the N-terminal region of eIF2β for its function in mammals.

scholarly journals Familial Parkinson's Disease Mutant E46K α-Synuclein Localizes to Membranous Structures, Forms Aggregates, and Induces Toxicity in Yeast Models

2011 ◽  
Vol 2011 ◽  
pp. 1-14 ◽  
Author(s):  
Michael Fiske ◽  
Michael White ◽  
Stephanie Valtierra ◽  
Sara Herrera ◽  
Keith Solvang ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Plasma Membrane ◽  
Mutant Form ◽  
Phospholipid Membrane ◽  
Endomembrane System ◽  
Wild Type ◽  
Significant Toxicity ◽  
Familial Parkinson’S Disease

In Parkinson’s disease (PD), midbrain dopaminergic neuronal death is linked to the accumulation of aggregated α-synuclein. The familial PD mutant form of α-synuclein, E46K, has not been thoroughly evaluated yet in an organismal model system. Here, we report that E46K resembled wild-type (WT) α-synuclein in Saccharomyces cerevisiae in that it predominantly localized to the plasma membrane, and it did not induce significant toxicity or accumulation. In contrast, in Schizosaccharomyces pombe, E46K did not associate with the plasma membrane. Instead, in one strain, it extensively aggregated in the cytoplasm and was as toxic as WT. Remarkably, in another strain, E46K extensively associated with the endomembrane system and was more toxic than WT. Our studies recapitulate and extend aggregation and phospholipid membrane association properties of E46K previously observed in vitro and cell culture. Furthermore, it supports the notion that E46K generates toxicity partly due to increased association with endomembrane systems within cells.

Abstract 14035: Renal Tubular Secretion and Cardiac Distribution of Dofetilide is Dependent on MATE1 Function

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Muhammad Erfan Uddin ◽  
Yan Jin ◽  
Alice A Gibson ◽  
Ingrid M Bonilla ◽  
Cynthia A Carnes ◽  
...  
Keyword(s):  
Organic Cation ◽  
Tubular Secretion ◽  
Hek293 Cells ◽  
Delayed Rectifier ◽  
Organic Cation Transporters ◽  
Wild Type ◽  
Renal Tubular Secretion ◽  
Cation Transporters ◽  
Renal Tubular

Introduction: Dofetilide is a delayed rectifier potassium channel inhibitor used to treat patients with atrial fibrillation and flutter, and its use is associated with a risk of QT prolongation and Torsades de Pointes . The mechanisms involved in dofetilide’s renal tubular secretion and its uptake into cardiomyocytes remain unknown. Previously reported drug-drug interaction (DDI) studies suggest the involvement of organic cation transporters. Here, we investigated the contribution of organic cation transporters (OCT2 and MATE1) to the pharmacokinetics of dofetilide to gain insight into its DDI potential. Hypothesis: Based on known DDIs with dofetilide, we hypothesize that OCT2 and/or MATE1 play a key role in the inter-individual variability in pharmacokinetics and pharmacodynamics of dofetilide. Methods: In vitro and ex vivo transport kinetics of dofetilide were determined in HEK293 cells stably transfected with OCT2 or MATE1, and in isolated cardiomyocytes, respectively. In vivo studies were performed in wild-type, OCT2-, and MATE1-deficient mice (n=5) receiving dofetilide (5 mg/kg, p.o., 2.5 mg/kg, i.v.), with or without several contraindicated drugs. Dofetilide concentrations in plasma and urine were determined by UPLC-MS/MS. Results: In vitro studies demonstrated that dofetilide is a good substrate of MATE1 but not OCT2. Deficiency of MATE1 was associated with increased plasma concentrations of dofetilide and with a significantly reduced urinary excretion (3-fold in females and 5-fold in males, respectively). Dofetilide accumulation in cardiomyocytes was increased by 2-fold in MATE1-deficient females, and pre-incubation with the MATE1 inhibitor cimetidine significantly reduced dofetilide uptake in wild-type cardiomyocytes. Several contraindicated drugs listed in the dofetilide prescribing information, including cimetidine, ketoconazole, increased dofetilide plasma exposure in wild-type mice by >2.8-fold. Conclusion: Renal secretion of dofetilide is mediated by MATE1 and is highly sensitive to inhibition by many widely used prescription drugs that can cause clinically relevant DDIs. Deficiency of MATE1 also increases accumulation in the heart which may contribute to individual variation in response to dofetilide.

Abstract 1503: Knockdown of Mog1 Expression Reduces Sodium Currents by Reducing the Trafficking of Cardiac Sodium Channel Na V 1.5 to the Cell Membrane

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Susmita Chakrabarti ◽  
Sandro Yong ◽  
Shin Yoo ◽  
Ling Wu ◽  
Qing Kenneth Wang
Keyword(s):  
Sodium Channel ◽  
Ventricular Myocytes ◽  
Sodium Current ◽  
Expression System ◽  
Heart Association ◽  
Hek293 Cells ◽  
Similar Reduction ◽  
Mammalian Expression ◽  
Ventricular Cells ◽  
Cardiac Sodium Channel

The cardiac sodium channel (Na v 1.5) plays a significant role in cardiac physiology and leads to cardiac arrhythmias and sudden death when mutated. Modulation of Na v 1.5 activity can also arise from changes to accessory subunits or proteins. Our laboratory has recently reported that MOG1, a small protein that is highly conserved from yeast to humans, is a co-factor of Na v 1.5. Increased MOG1 expression has been shown to increase Na v 1.5 current density. In adult mouse ventricular myocytes, these two proteins were found to be co-localized at the intercalated discs. Here, we further characterize the regulatory role of MOG1 using the RNA interference technique. Sodium current was recorded in voltage-clamp mode from a holding potential of −100 mV and activated to −20 mV. In 3-day old mouse neonatal ventricular cells transfected with siRNA against mouse MOG1 decreased sodium current densities (pA/pF) compared to control or scramble siRNA treated cells (−10.2±3.3, n=11 vs. −165±16, n=20 or −117.9±11.7, n=11). A similar reduction in sodium current was observed in mammalian expression system consisting of HEK293 cells stably expressing human Na v 1.5, by transfecting siRNAs against either human or mouse MOG1 (−41.7±8.3, n=7 or, −82.6±9.6, n=7 vs. −130.6±11.5, n=7; −111.5±8.5, n=7, respectively). Immunocytochemistry revealed that the expression of MOG1 and Na v 1.5 were decreased in both HEK and neonatal cells when compared to scramble siRNAs or control groups. These results show that MOG1 is an essential co-factor for Na v 1.5 by way of a channel trafficking. Such interactions between MOG1 and Na v 1.5 suggest that early localization of MOG1 on the membrane of neonatal cardiomyocytes may be necessary for proper localization and the distribution of Na v 1.5 during cardiac development. This research has received full or partial funding support from the American Heart Association, AHA National Center.

scholarly journals Angiogenic Deficiency and Adipose Tissue Dysfunction Are Associated with Macrophage Malfunction in SIRT1−/− Mice

Endocrinology ◽  
2012 ◽  
Vol 153 (4) ◽  
pp. 1706-1716 ◽  
Author(s):  
Fen Xu ◽  
David Burk ◽  
Zhanguo Gao ◽  
Jun Yin ◽  
Xia Zhang ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
Adipocyte Differentiation ◽  
Nuclear Factor Κb ◽  
The Body ◽  
Sirtuin 1 ◽  
Vascular Density ◽  
Peroxisome Proliferator ◽  
Wild Type ◽  
Peroxisome Proliferator Activated Receptor

The histone deacetylase sirtuin 1 (SIRT1) inhibits adipocyte differentiation and suppresses inflammation by targeting the transcription factors peroxisome proliferator-activated receptor γ and nuclear factor κB. Although this suggests that adiposity and inflammation should be enhanced when SIRT1 activity is inactivated in the body, this hypothesis has not been tested in SIRT1 null (SIRT1−/−) mice. In this study, we addressed this issue by investigating the adipose tissue in SIRT1−/− mice. Compared with their wild-type littermates, SIRT1 null mice exhibited a significant reduction in body weight. In adipose tissue, the average size of adipocytes was smaller, the content of extracellular matrix was lower, adiponectin and leptin were expressed at 60% of normal level, and adipocyte differentiation was reduced. All of these changes were observed with a 50% reduction in capillary density that was determined using a three-dimensional imaging technique. Except for vascular endothelial growth factor, the expression of several angiogenic factors (Pdgf, Hgf, endothelin, apelin, and Tgf-β) was reduced by about 50%. Macrophage infiltration and inflammatory cytokine expression were 70% less in the adipose tissue of null mice and macrophage differentiation was significantly inhibited in SIRT1−/− mouse embryonic fibroblasts in vitro. In wild-type mice, macrophage deletion led to a reduction in vascular density. These data suggest that SIRT1 controls adipose tissue function through regulation of angiogenesis, whose deficiency is associated with macrophage malfunction in SIRT1−/− mice. The study supports the concept that inflammation regulates angiogenesis in the adipose tissue.

scholarly journals ThePDE1-encoded Low-Affinity Phosphodiesterase in the YeastSaccharomyces cerevisiaeHas a Specific Function in Controlling Agonist-induced cAMP Signaling

1999 ◽  
Vol 10 (1) ◽  
pp. 91-104 ◽  
Author(s):  
Pingsheng Ma ◽  
Stefaan Wera ◽  
Patrick Van Dijck ◽  
Johan M. Thevelein
Keyword(s):  
Feedback Inhibition ◽  
Phosphorylation Site ◽  
Camp Signaling ◽  
Intrinsic Activity ◽  
Camp Accumulation ◽  
Wild Type ◽  
Intracellular Acidification ◽  
Crude Extracts

The yeast Saccharomyces cerevisiae contains two genes, PDE1 and PDE2, which respectively encode a low-affinity and a high-affinity cAMP phosphodiesterase. The physiological function of the low-affinity enzyme Pde1 is unclear. We show that deletion of PDE1, but not PDE2, results in a much higher cAMP accumulation upon addition of glucose or upon intracellular acidification. Overexpression of PDE1, but not PDE2, abolished the agonist-induced cAMP increases. These results indicate a specific role for Pde1 in controlling glucose and intracellular acidification-induced cAMP signaling. Elimination of a putative protein kinase A (PKA) phosphorylation site by mutagenesis of serine252into alanine resulted in a Pde1ala252allele that apparently had reduced activity in vivo. Its presence in a wild-type strain partially enhanced the agonist-induced cAMP increases compared with pde1Δ. The difference between the Pde1ala252allele and wild-type Pde1 was strongly dependent on PKA activity. In a RAS2val19pde2Δ background, the Pde1ala252allele caused nearly the same hyperaccumulation of cAMP as pde1Δ, while its expression in a PKA-attenuated strain caused the same reduction in cAMP hyperaccumulation as wild-type Pde1. These results suggest that serine252might be the first target site for feedback inhibition of cAMP accumulation by PKA. We show that Pde1 is rapidly phosphorylated in vivo upon addition of glucose to glycerol-grown cells, and this activation is absent in the Pde1ala252mutant. Pde1 belongs to a separate class of phosphodiesterases and is the first member shown to be phosphorylated. However, in vitro the Pde1ala252enzyme had the same catalytic activity as wild-type Pde1, both in crude extracts and after extensive purification. This indicates that the effects of the S252A mutation are not caused by simple inactivation of the enzyme. In vitro phosphorylation of Pde1 resulted in a modest and variable increase in activity, but only in crude extracts. This was absent in Pde1ala252, and phosphate incorporation was strongly reduced. Apparently, phosphorylation of Pde1 does not change its intrinsic activity or affinity for cAMP but appears to be important in vivo for protein-protein interaction or for targeting Pde1 to a specific subcellular location. The PKA recognition site is conserved in the corresponding region of the Schizosaccharomyces pombe and Candida albicans Pde1 homologues, possibly indicating a similar control by phosphorylation.

scholarly journals Differential phosphorylation signals control endocytosis of GPR15

2017 ◽  
Vol 28 (17) ◽  
pp. 2267-2281 ◽  
Author(s):  
Yukari Okamoto ◽  
Sojin Shikano
Keyword(s):  
Surface Density ◽  
Functional Role ◽  
Control Cell ◽  
Phosphorylation Site ◽  
Hek293 Cells ◽  
C Terminus ◽  
Differential Phosphorylation ◽  
G Protein Coupled

GPR15 is an orphan G protein–coupled receptor (GPCR) that serves for an HIV coreceptor and was also recently found as a novel homing receptor for T-cells implicated in colitis. We show that GPR15 undergoes a constitutive endocytosis in the absence of ligand. The endocytosis was clathrin dependent and partially dependent on β-arrestin in HEK293 cells, and nearly half of the internalized GPR15 receptors were recycled to the plasma membrane. An Ala mutation of the distal C-terminal Arg-354 or Ser-357, which forms a consensus phosphorylation site for basophilic kinases, markedly reduced the endocytosis, whereas phosphomimetic mutation of Ser-357 to Asp did not. Ser-357 was phosphorylated in vitro by multiple kinases, including PKA and PKC, and pharmacological activation of these kinases enhanced both phosphorylation of Ser-357 and endocytosis of GPR15. These results suggested that Ser-357 phosphorylation critically controls the ligand-independent endocytosis of GPR15. The functional role of Ser-357 in endocytosis was distinct from that of a conserved Ser/Thr cluster in the more proximal C-terminus, which was responsible for the β-arrestin– and GPCR kinase–dependent endocytosis of GPR15. Thus phosphorylation signals may differentially control cell surface density of GPR15 through endocytosis.

Glycosylation of the osmoresponsive transient receptor potential channel TRPV4 on Asn-651 influences membrane trafficking

2006 ◽  
Vol 290 (5) ◽  
pp. F1103-F1109 ◽  
Author(s):  
Hongshi Xu ◽  
Yi Fu ◽  
Wei Tian ◽  
David M. Cohen
Keyword(s):  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Transient Receptor Potential ◽  
Receptor Potential ◽  
Trp Channel ◽  
Hek293 Cells ◽  
Wild Type ◽  
Functional Feature ◽  
Voltage Gated ◽  
Transient Receptor

We identified a consensus N-linked glycosylation motif within the pore-forming loop between the fifth and sixth transmembrane segments of the osmoresponsive transient receptor potential (TRP) channel TRPV4. Mutation of this residue from Asn to Gln (i.e., TRPV4N651Q) resulted in loss of a slower migrating band on anti-TRPV4 immunoblots and a marked reduction in lectin-precipitable TRPV4 immunoreactivity. HEK293 cells transiently transfected with the mutant TRPV4N651Q exhibited increased calcium entry in response to hypotonic stress relative to wild-type TRPV4 transfectants. This increase in hypotonicity responsiveness was associated with an increase in plasma membrane targeting of TRPV4N651Q relative to wild-type TRPV4 in both HEK293 and COS-7 cells but had no effect on overall channel abundance in whole cell lysates. Residue N651 of TRPV4 is immediately adjacent to the pore-forming loop. Although glycosylation in this vicinity has not been reported for a TRP channel, the structurally related hexahelical hyperpolarization-activated cyclic nucleotide-gated channel, HCN2, and the voltage-gated potassium channel, human ether-a-go-go-related (HERG), share a nearly identically situated and experimentally confirmed N-linked glycosylation site which promotes rather than limits channel insertion into the plasma membrane. These data point to a potentially conserved structural and functional feature influencing membrane trafficking across diverse members of the voltage-gated-like ion channel superfamily.

Phosphorylation of threonine 276 in Smad4 is involved in transforming growth factor-β-induced nuclear accumulation

2003 ◽  
Vol 285 (4) ◽  
pp. C823-C830 ◽  
Author(s):  
Bernard A. J. Roelen ◽  
Ori S. Cohen ◽  
Malay K. Raychowdhury ◽  
Deborah N. Chadee ◽  
Ying Zhang ◽  
...  
Keyword(s):  
Growth Factor ◽  
Transforming Growth Factor ◽  
Phosphorylation Site ◽  
Transforming Growth Factor Β ◽  
Nuclear Accumulation ◽  
Mutant Protein ◽  
Wild Type ◽  
Type Protein ◽  
Wild Type Protein

Smad4, the common Smad, is central for transforming growth factor (TGF)-β superfamily ligand signaling. Smad4 has been shown to be constitutively phosphorylated (Nakao A, Imamura T, Souchelnytskyi S, Kawabata M, Ishisaki A, Oeda E, Tamaki K, Hanai J, Heldin C-H, Miyazono K, and ten Dijke P. EMBO J 16: 5353-5362, 1997), but the site(s) of phosphorylation, the kinase(s) that performs this phosphorylation, and the significance of the phosphorylation of Smad4 are currently unknown. This report describes the identification of a consensus ERK phosphorylation site in the linker region of Smad4 at Thr276. Our data show that ERK can phosphorylate Smad4 in vitro but not Smad4 with mutated Thr276. Flag-tagged Smad4-T276A mutant protein accumulates less efficiently in the nucleus after stimulation by TGF-β and is less efficient in generating a transcriptional response than Smad4 wild-type protein. Tryptic phosphopeptide mapping identified a phosphopeptide in Smad4 wild-type protein that was absent in phosphorylated Smad4-T276A mutant protein. Our results suggest that MAP kinase can phosphorylate Thr276 of Smad4 and that phosphorylation can lead to enhanced TGF-β-induced nuclear accumulation and, as a consequence, enhanced transcriptional activity of Smad4.

scholarly journals Single Cell RNA-Seq Characterises Pre-Leukemic Transformation Driven By CEBPA N321D in the Hoxb8-FL Cell Line

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3887-3887
Author(s):  
Moosa Qureshi ◽  
Fernando Calero-Nieto ◽  
Iwo Kucinski ◽  
Sarah Kinston ◽  
George Giotopoulos ◽  
...  
Keyword(s):  
Dendritic Cell ◽  
Single Cell ◽  
Cell Line ◽  
Single Cells ◽  
Mutant Form ◽  
Rna Seq ◽  
Wild Type ◽  
Leukemic Transformation

Abstract The C/EBPα transcription factor plays a pivotal role in myeloid differentiation and E2F-mediated cell cycle regulation. Although CEBPA mutations are common in acute myeloid leukaemia (AML), little is known regarding pre-leukemic alterations caused by mutated CEBPA. Here, we investigated early events involved in pre-leukemic transformation driven by CEBPA N321D in the LMPP-like cell line Hoxb8-FL (Redecke et al., Nat Methods 2013), which can be maintained in vitro as a self-renewing LMPP population using Flt3L and estradiol, as well as differentiated both in vitro and in vivo into myeloid and lymphoid cell types. Hoxb8-FL cells were retrovirally transduced with Empty Vector (EV), wild-type CEBPA (CEBPA WT) or its N321D mutant form (CEBPA N321D). CEBPA WT-transduced cells showed increased expression of cd11b and SIRPα and downregulation of c-kit, suggesting that wild-type CEBPA was sufficient to promote differentiation even under LMPP growth conditions. Interestingly, we did not observe the same phenotype in CEBPA N321D-transduced cells. Upon withdrawal of estradiol, both EV and CEBPA WT-transduced cells differentiated rapidly into a conventional dendritic cell (cDC) phenotype by day 7 and died within 12 days. By contrast, CEBPA N321D-transduced cells continued to grow for in excess of 56 days, with an initial cDC phenotype but by day 30 demonstrating a plasmacytoid dendritic cell precursor phenotype. CEBPA N321D-transduced cells were morphologically distinct from EV-transduced cells. To test leukemogenic potential in vivo, we performed transplantation experiments in lethally irradiated mice. Serial monitoring of peripheral blood demonstrated that Hoxb8-FL derived cells had disappeared by 4 weeks, and did not reappear. However, at 6 months CEBPA N321D-transduced cells could still be detected in bone marrow in contrast to EV-transduced cells but without any leukemic phenotype. To identify early events involved in pre-leukemic transformation, the differentiation profiles of EV, CEBPA WT and CEBPA N321D-transduced cells were examined with single cell RNA-seq (scRNA-seq). 576 single cells were taken from 3 biological replicates at days 0 and 5 post-differentiation, and analysed using the Automated Single-Cell Analysis Pipeline (Gardeux et al., Bioinformatics 2017). Visualisation by t-SNE (Fig 1) demonstrated: (i) CEBPA WT-transduced cells formed a distinct cluster at day 0 before withdrawal of estradiol; (ii) CEBPA N321D-transduced cells separated from EV and CEBPA WT-transduced cells after 5 days of differentiation, (iii) two subpopulations could be identified within the CEBPA N321D-transduced cells at day 5, with a cluster of five CEBPA N321D-transduced single cells distributed amongst or very close to the day 0 non-differentiated cells. Differential expression analysis identified 224 genes upregulated and 633 genes downregulated specifically in the CEBPA N321D-transduced cells when compared to EV cells after 5 days of differentiation. This gene expression signature revealed that CEBPA N321D-transduced cells switched on a HSC/MEP/CMP transcriptional program and switched off a myeloid dendritic cell program. Finally, in order to further dissect the effect of the N321D mutation, the binding profile of endogenous and CEBPA N321D was compared by ChIP-seq before and after 5 days of differentiation. Integration with scRNA-seq data identified 160 genes specifically downregulated in CEBPA N321D-transduced cells which were associated with the binding of the mutant protein. This list of genes included genes previously implicated in dendritic cell differentiation (such as NOTCH2, JAK2), as well as a number of genes not previously implicated in the evolution of AML, representing potentially novel therapeutic targets. Disclosures No relevant conflicts of interest to declare.

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