Human Erythroleukemia Cells Research Articles

Synthesis of Flavone Derivatives via N-Amination and Evaluation of Their Anticancer Activities.

Seventeen new flavone derivatives substituted at the 4′-OH position were designed, synthesized and evaluated for their anticancer and antibacterial activities. Among them, compounds 3, 4, 6f, 6e, 6b, 6c and 6k demonstrated the most potent antiproliferative activities against a human erythroleukemia cell line (HEL) and a prostate cancer cell line (PC3). The results also showed that the IC50 value of compounds 3, 4, 6f, 6e, 6b, 6c and 6k were close to that of the anticancer drug cisplatin (DDP) and lower than that of apigenin. All of the derivatives did not present antibacterial activities. The structure–activity relationships evaluation showed that the configuration of methyl amino acid might affect their biological activities.

Linoleic acid-rich guava seed oil: Safety and bioactivity.

Guava (Psidium guajava) is a widely consumed fruit and has been commercialized in markets. The seeds are by-products of the processing procedures performed by the commercial guava juice industry. They are considered a nutritional resource that has been poorly utilized as they contain essential fatty acids such as linoleic acid (LA) and phenolics in abundance. In the study, guava seed oil (GSO) was used, which was obtained by hexane extraction of guava seeds to determine composition and test toxicity, cell migration, cancer cell viability, and plasmodium growth. GSO was found to be relatively nontoxic to normal hepatocytes and peripheral blood mononuclear cells, with mice for 14days showing median lethal dose (LD50 )>10mg/kg and rats for up to 90days. Surprisingly, the oil inhibited the proliferation of the human erythroleukemic cells in a dose-dependent manner with the half maximal inhibitory concentration values of 155 and 137μg/ml at 24 and 48hr, respectively. Importantly, GSO at 500μg/ml was found to increase the degree of migration of keratinocytes (HaCaT). These observations suggest that edible P.guajava seed oil, which is abundant with linoleic acid and antioxidants, can promote skin wound healing and inhibit the proliferation of leukemic cells.

A Human Long Non-coding RNA LncATV Promotes Virus Replication Through Restricting RIG-I-Mediated Innate Immunity.

Pattern recognition receptors sense pathogen components and initiate the host antiviral innate immune response, such as inducing interferons (IFNs). Long non-coding RNAs (lncRNAs) are emerging regulators of multiple biological processes. However, their role in antiviral response, especially through regulating the human innate immune, is largely unexplored. Here we characterized that lncATV, a human specific lncRNA, was up-regulated upon type I/III IFN stimulations and virus infection. LncATV was cytoplasmic localized and relatively high expressed in human monocytes, erythroleukemia cells and hepatoma cells. Notably, lncATV knockdown significantly inhibited the replication of multiple RNA viruses, such as hepatitis C virus, Zika virus, Newcastle disease virus, and Sendai virus. Mechanistically, RIG-I antiviral signaling and IFN effective pathway were enhanced when lncATV expression was knocked down but inhibited by overexpressed lncATV. RNA immunoprecipitation results demonstrated an association between LncATV and RIG-I. Collectively, our findings reveal the functional role of a novel human specific lncATV as a regulatory lncRNA restricting virus associated innate immune response.

Inflammasome Regulates Hematopoiesis through Cleavage of the Master Erythroid Transcription Factor GATA1

Chronic inflammatory diseases are associated with altered hematopoiesis that could result in neutrophilia and anemia. Here we report that genetic or chemical manipulation of different inflammasome components altered the differentiation of hematopoietic stem and progenitor cells (HSPC) in zebrafish. Although the inflammasome was dispensablefor the emergence of HSPC, it was intrinsically required for their myeloid differentiation. In addition, Gata1 transcript and protein amounts increased in inflammasome-deficient larvae, enforcing erythropoiesis and inhibiting myelopoiesis. This mechanism is evolutionarily conserved, since pharmacological inhibition of the inflammasome altered erythroid differentiation of human erythroleukemic K562 cells. In addition, caspase-1 inhibition rapidly upregulated GATA1 protein in mouse HSPC promoting their erythroid differentiation. Importantly, pharmacological inhibition of the inflammasome rescued zebrafish disease models of neutrophilic inflammation and anemia. These results indicate that the inflammasome plays a major role in the pathogenesis of neutrophilia and anemia of chronic diseases and reveal druggable targets for therapeutic interventions.

Organometallic rhenium tricarbonyl-enrofloxacin and -levofloxacin complexes: synthesis, albumin-binding, DNA-interaction and cell viability studies.

Organometallic rhenium complexes have recently been considered in the development of novel antitumor agents due to their suitable properties. A series of rhenium(I) tricarbonyl complexes was synthesized with the quinolone antimicrobial agents enrofloxacin (Herx) and levofloxacin (Hlfx) and solvent (e.g., methanol), imidazole (im) or pyridine (py) as co-ligands. The complexes were characterized by spectroscopic methods. The interaction of the rhenium complexes with bovine serum albumin was investigated by fluorescence emission spectroscopy and the corresponding binding constants were determined. The binding of the rhenium complexes to calf-thymus DNA was monitored by UV-Vis spectroscopy, viscosity measurements and competitive studies with ethidium bromide. These studies indicated that intercalation is the most possible mode of action and the corresponding DNA-binding constants of the complexes were calculated. The cytotoxicity of the Re-complexes against human K-562 erythroleukemia cells was found to be moderate to high. These preliminary results are promising and warrant the design of new Re-complexes with improved properties in future studies.

Thiourea derivatives induce fetal hemoglobin production in-vitro: A new class of potential therapeutic agents for β-thalassemia

Fetal hemoglobin (HbF) induction is a cost-effective therapeutic approach for the treatment of β-hemoglobinopathies like β-thalassemia and sickle cell anemia. The present study discusses the potential of thiourea derivatives as new class of compounds that induce the fetal hemoglobin production. HbF inducing effect of thiourea derivatives was studied using experimental cell system, the human erythroleukemic K562 cell line. Erythroid induction of K562 cells was studied by the benzidine/H2O2 reaction, total hemoglobin production was estimated by plasma hemoglobin assay kit, and γ-globin gene expression by RT-qPCR, whereas fetal hemoglobin production was estimated by flow cytometry and immunofluorescence microscopy. The results indicated that newly synthesized thiourea derivative are potent inducers of erythroid differentiation of K562 cells with an increased γ-globin gene expression and fetal hemoglobin production. Moreover, these compounds showed no cytotoxic effect and inhibition on K562 cells at HbF inducing concentrations. It is important to note that hydroxyurea is a cytotoxic chemotherapeutic agent and have deleterious side effects, reflecting the need to identify new safe and effective HbF induces. These results signify thiourea derivatives as promising HbF inducers, with the potential to be studied against hematological disorders, including β-thalassemia and sickle cell anemia.

Design, synthesis, and cytotoxic activities of novel hybrids of parthenolide and thiazolidinedione via click chemistry

A series of novel parthenolide–thiazolidinedione hybrids have been synthesized via a click chemistry-mediated coupling between parthenolide and thiazolidinedione, and evaluated for their cytotoxic activities. The results indicated that all the hybrids showed moderate cytotoxic effects on human cancer cell lines, including human erythroleukemia cell line (HEL), prostate (PC3), and breast (MDA-MB-231) by MTT assay. In particular, compound VI-6 exhibited the best cytotoxic activities against the MDA-MB-231 cells with IC50 value of 2.07 µM, which was about eight times more active than that of the original compound (PTL). These interesting results might be used to develop novel lead scaffolds for potential anticancer agents.

A major interspecies difference in the ionic selectivity of megakaryocyte Ca2+-activated channels sensitive to the TMEM16F inhibitor CaCCinh-A01

TMEM16F is a surface membrane protein critical for platelet procoagulant activity, which exhibits both phospholipid scramblase and ion channel activities following sustained elevation of cytosolic Ca2+. The extent to which the ionic permeability of TMEM16F is important for platelet scramblase responses remains controversial. To date, only one study has reported the electrophysiological properties of TMEM16F in cells of platelet/megakaryocyte lineage, which observed cation-selectivity within excised patch recordings from murine marrow-derived megakaryocytes. This contrasts with reports using whole-cell recordings that describe this channel as displaying either selectivity for anions or being relatively non-selective amongst the major physiological monovalent ions.We have studied TMEM16F expression and channel activity in primary rat and mouse megakaryocytes and the human erythroleukemic (HEL) cell line that exhibits megakaryocytic surface markers. Immunocytochemical analysis was consistent with surface TMEM16F expression in cells from all three species. Whole-cell recordings in the absence of K+-selective currents revealed an outwardly rectifying conductance activated by a high intracellular Ca2+ concentration in all three species. These currents appeared after 5–6 minutes and were blocked by CaCCinh-A01, properties typical of TMEM16F. Ion substitution experiments showed that the underlying conductance was predominantly Cl–-permeable in rat megakaryocytes and HEL cells, yet non-selective between monovalent anions and cations in mouse megakaryocytes. In conclusion, the present study further highlights the difference in ionic selectivity of TMEM16F in platelet lineage cells of the mouse compared to other mammalian species. This provides additional support for the ionic “leak” hypothesis that the scramblase activity of TMEM16F does not rely upon its ability to conduct ions of a specific type.

Design, synthesis and structure-activity relationship study of aminopyridine derivatives as novel inhibitors of Janus kinase 2

Janus Kinase 2 (JAK2) is a kind of intracellular non-receptor protein tyrosine kinase and has been certified as an important target for the treatment of myeloproliferative neoplasms and rheumatoid arthritis. However, the low selectivity and potential safety issues restrict the clinical applications of JAK2 inhibitors. Here we found that crizotinib showed good inhibitory activity against JAK2 by enzymatic assays (IC50 = 27 nM). Then we carried out structure-based drug design and synthesized a series of compounds with an aminopyridine scaffold. Finally, compound 12k and 12l were identified as the promising inhibitors of JAK2, which exhibited high inhibitory activity (IC50 = 6 nM and 3 nM, respectively) and selectivity for JAK2 over JAK1 and JAK3, and showed potent antiproliferative activities toward HEL human erythroleukemia cells. Moreover, 12k suppressed symptoms of the collagen-induced arthritis (CIA) model in rats.

Cell Growth Inhibition and Transglutaminase 2 Expression in Human Erythroleukemia Cells (K562) Following Exposure to Retinoic Acid and Sodium Butyrate

Transglutaminase (TG‐2) is a calcium‐dependent enzyme that has been linked to numerous biological functions including cell adhesion, extracellular matrix, stabilization, wound healing, receptor signaling, cellular proliferation, and cellular motility. This enzyme is also thought to play a role in cancer, as TG‐2 expression is elevated in several types of cancer cells. Some studies have also shown that patients with neurodegenerative diseases like Parkinson's, and Alzheimer's have exhibited elevated levels of TG‐2 which may be linked to the formation of protein aggregates that cause these diseases. The goal of this study was to explore the biological responses of human erythroleukemia cells (K562) to retinoic acid and sodium butyrate. The cells were treated with DMSO (20 uL), sodium butyrate (100 uM), retinoic acid (20 uM), or a combination of sodium butyrate (100 uM) and retinoic acid (20 uM). Preliminary data has shown that TG‐2 is expressed more in cells treated with sodium butyrate, while retinoic acid does not have an effect on TG‐2 expression. The control cells grew continually over the 72‐hour observation period, while the growth of cells treated with sodium butyrate and the mix of both chemicals were slowed to approximately 50% of the growth of control cells. Immunofluorescent photo‐microscopy was used to measure the expression of TG‐2. The cells were also tested for apoptosis using fluorescent microscopy to differentiate between viable, necrotic, and apoptotic cells. Future research will attempt to localize the actual location of the TG‐2 when treated with both chemicals, and if it promotes or suppresses the viability of the cancer cells. Studies are also underway to determine the amount of TG‐2 that is present in each of the samples.Support or Funding InformationFunding provided by Department of Chemistry, Slippery Rock UniversityThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cell Growth and Transglutaminase 2 Expression within the Human Erythroleukemia Cell Line (HEL)

The objective of the study was to explore the biological responses of retinoic acid and sodium butyrate on human erythroleukemia cells (HEL). The process of apoptosis is exemplary in that the cytoskeleton collapses and the DNA is broken into fragments. The cellular components of the dead cell after undergoing apoptosis are able to be recycled. Furthermore, a cell that has experienced apoptosis does not disturb its neighbors. By learning the basics of human cell experimentation, further analysis was conducted of the HEL cell lines, such as an apoptotic assay and indirect immunofluorescence. Cells were treated with either buffer (control), retinoic acid (20 uM), sodium butyrate (100 uM) or a combination of retinoic acid (20 uM) and sodium butyrate (100 uM). Cell growth rates declined when the cells were treated with the chemicals; the greatest difference being in the combination culture of retinoic acid and sodium butyrate. The enzyme transglutaminase (TG2) is an enzyme found in large amounts within some cancer cells, and is known to have multiple functions. When the cells were treated with a monoclonal antibody to TG2, transglutaminase enzyme fluorescence in the control cultures were observed to be cytoplasmic. The retinoic acid‐treated cultures appeared to have a strong presence of the enzyme in the cytoplasm. The amount of TG2 appeared to increase in all the cultures with time with the greatest increase occurring in the combination cultures. After 144 hours a portion of the combination cultures had been converted to apoptotic bodies. Future research will attempt to localize the actual location of the transglutaminase when treated with both chemicals, and if it promotes or suppresses the viability of the cancer cells. Studies are underway to determine the amount of transglutaminase that is present in each of the samples.Support or Funding InformationFunding provided by Department of Chemistry, Slippery Rock UniversityThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Calcium Dependence of Transglutaminase and its Function in Apoptosis in Human Erythroleukemia Cell Lines

The expression of transglutaminase (TG‐2) in human erythroleukemia cells is dependent on the intracellular calcium concentration. Human erythroleukemia cells (K562) arise from stem cells that have not fully differentiated to erythrocytes and maintain continuous, uncontrolled proliferation. The preferred apoptotic machinery reduces the degree of inflammation of the tumor. Previous studies have concluded that thapsigargin induces the rate of apoptosis in these cells and disrupts the expressivity of TG‐2. Expression of the TG‐2 protein can be visualized when complexed with CUB74, a monoclonal antibody to TG‐2, and GAM‐FITC. Immunofluorescent studies suggest that thapsigargin and an increase in available Ca2+ upregulates the expression of TG‐2 type proteins. Cells exposed to calcium shock also demonstrated an increase in the presence of TG‐2 on the periphery and intrarnuclear space of the cell. Calcium shocking cells may open the nuclear envelope to allow access of TG‐2, or stimulates the synthesis of the protein coding gene. EDTA, a popular chelating agent and DNase repressor, reduces the expression of TG‐2. The mechanism for this remains elusive. Investigations will be conducted to visualize any change in the amount of mRNA product as well as quantifying the difference in TG‐2 products.Support or Funding InformationFunding provided by Department of Chemistry, Slippery Rock UniversityThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

MS-275 Chemical Analogues Promote Hemoglobin Production and Erythroid Differentiation of K562 Cells

β-Thalassemia (β-thal) is a hemoglobinopathy characterized by reduced or absent β-globin production. Pharmacological reactivation of the γ-globin gene for the production of fetal hemoglobin (Hb F) presents an attractive treatment strategy. In an effort to identify promising therapeutic agents, we evaluated 80 analogues of the histone deacetylase inhibitor MS-275, a known Hb F inducer. The chemical analogues were identified via molecular modeling and targeted chemical modifications. Nine novel agents exhibited significant hemoglobin (Hb)-inducing and erythroid differentiation activities in the human K562 erythroleukemia cell line. Five of them appeared to be stronger inducers than the lead compound, MS-275, demonstrating the effectiveness of our method.

Di-O-lauroyl-decitabine-lipid nanocapsules: toward extending decitabine activity.

BackgroundAcute myeloid leukemia mainly affects adult patients. Complete remission for patients younger than 60 years, who are candidates for standard induction therapy, is achieved in 60%–80% of cases. However, the prognosis is still poor for older patients, who are unfit for intensive chemotherapy, and only a few therapies are available. Hypomethylating agents, such as decitabine, are approved for such patients. The current dosing regimen consists of one administration per day, for 5 days, each 4 weeks.MethodsHere, we present the synthesis of a decitabine prodrug, combined with its encapsulation into a lipid-based nanocapsule formulation. Decitabine (C12)2 was synthetized, then loaded into nanocapsules. Its stability in phosphate buffer ans human plasma was checked. Its activity was evaluated by Cell proliferation assays and cell-cycle analysis on human erythroleukemia cells. Then its pharmacokinetics was determined on a rat model.ResultsDecitabine (C12)2 was obtained with a yield of 50%. Drug loading into nanocarriers of 27.45±0.05 nm was 5.8±0.5 mg/mL. The stability of decitabine was improved and its activity on leukemia cells was not altered. Finally, pharmacokinetics studies showed a prolonged mean residence time of the drug.ConclusionDecitabine (C12)2 as a prodrug showed high encapsulation efficiency, a good stability in plasma with no impact on its activity on leukemia cells and improved pharmacokinetics.

Undergraduate lab series using the K562 human leukemia cell line: Model for cell growth, death, and differentiation in an advanced cell biology course.

This sequence of labs was developed for an upper level undergraduate cell biology course at Fairfield University. The labs are based on the use of the K562 human erythroleukemia cell line, a model system that is exceptionally amenable to an undergraduate cell biology lab course due to its ease of maintenance and propagation and usefulness for studies of growth, death, and differentiation. The sequence of labs is conducted over a 6-week period, following a series of weekly cell biology labs covering basic cell and molecular biology techniques. Together, the lab series has four primary objectives 1) to teach students how to culture and maintain mammalian cells; 2) to build student competency in standard cell biology techniques; 3) to demonstrate the role of growth factors on cell proliferation and viability; and 4) to provide students with an opportunity to use these cells in an independent investigation on cell differentiation. We provide examples of student data and offer a range of experimental measurements depending on institutional capacity and facilities. Our assessment data suggest that students find great value in this lab series, enhancing their comprehension of key concepts, acquisition of important lab skills, and depth of understanding of the research process. © 2019 International Union of Biochemistry and Molecular Biology, 47(3):263-271, 2019.

Discovery and evaluation of ZT55, a novel highly-selective tyrosine kinase inhibitor of JAK2V617F against myeloproliferative neoplasms

BackgroundThe JAK2-STAT signaling pathway plays a critical role in myeloproliferative neoplasms (MPN). An activating mutation in JAK2 (V617F) is present in ~ 95% of polycythemia vera, essential thrombocythemia, and primary myelofibrosis cases. This study aims to explore the selective JAK2V617F inhibitor, evaluate the efficacy and possible mechanism of ZT55 on MPN.MethodsHTRF assays were conducted to evaluate the selective inhibition of ZT55 for JAKs. Cell apoptosis, proliferation, and cycle arrest assays were performed to examine the effect of ZT55 on HEL cell line with JAK2V617F mutation in vitro. Western analysis was used to monitor the expression and activity of proteins on JAK2/STAT pathway. A mice xenograft model was established to evaluate the antitumor efficacy of ZT55 in vivo. Peripheral blood samples from patients with the JAK2V617F mutation were collected to estimate the effect of ZT55 on erythroid colony formation by colony-forming assay.ResultsWe found that ZT55 showed a selective inhibition of a 0.031 μM IC50 value against JAK2. It exhibited potent effects on the cellular JAK-STAT pathway, inhibiting tyrosine phosphorylation in JAK2V617F and downstream STAT3/5 transcription factors. ZT55 inhibited the proliferation of the JAK2V617F-expressing HEL cell line, leading to cell cycle arrest at the G2/M phase and induction of caspase-dependent apoptosis. Notably, ZT55 also significantly suppressed the growth of HEL xenograft tumors in vivo. Further evaluation indicated that ZT55 blocked erythroid colony formation of peripheral blood hematopoietic progenitors from patients carrying the JAK2V617F mutation.ConclusionThese results suggest that ZT55 is a highly-selective JAK2 inhibitor that can induce apoptosis of human erythroleukemia cells by inhibiting the JAK2-STAT signaling.

IL-6 stimulation of DNA replication is JAK1/2 mediated in cross-talk with hyperactivated ERK1/2 signaling.

Myeloproliferative neoplasms (MPNs) are developing resistance to therapy by JAK1/2 inhibitor ruxolitinib. To explore the mechanism of ruxolitinib's limited effect, we examined the JAK1/2 mediated induction of proliferation related ERK1/2 and AKT signaling by proinflammatory interleukin-6 (IL-6) in MPN granulocytes and JAK2V617F mutated human erythroleukemia (HEL) cells. We found that JAK1/2 or JAK2 inhibition prevented the IL-6 activation of STAT3 and AKT pathways in polycythemia vera and HEL cells. Further, we showed that these inhibitors also blocked the IL-6 activation of the AKT pathway in primary myelofibrosis (PMF). Only JAK1/2 inhibitor ruxolitinib largely activated ERK1/2 signaling in essential thrombocythemia and PMF (up to 4.6 fold), with a more prominent activation in JAK2V617F positive granulocytes. Regarding a cell cycle, we found that IL-6 reduction of HEL cells percentage in G2M phase was reversed by ruxolitinib (2.6 fold). Moreover, ruxolitinib potentiated apoptosis of PMF granulocytes (1.6 fold). Regarding DNA replication, we found that ruxolitinib prevented the IL-6 augmentation of MPN granulocytes frequency in the S phase of the cell cycle (up to 2.9 fold). The inflammatory stimulation induces a cross-talk between the proliferation linked pathways, where JAK1/2 inhibition is compensated by the activation of the ERK1/2 pathway during IL-6 stimulation of DNA replication.

Cinchona alkaloids as natural fetal hemoglobin inducing agents in human erythroleukemia cells

Pharmacologically mediated reactivation of γ-globin gene with an increase in fetal hemoglobin production, is a cost effective experimental therapeutic intervention for the management of β-hemoglobinopathies. Investigation of new pharmacological agents as HbF inducers from natural resources is desirable to develop safe and effective HbF inducers. We evaluated selected cinchona alkaloids (cinchonidine and quinidine) for their potential of erythroid differentiation and augmentation of fetal hemoglobin production. K562 cells were used as in vitro experimental model. Erythroid differentiation of K562 cells was studied using a benzidine assay, and total hemoglobin was estimated through a calorimetric method. Whereas, quantitative real-time PCR (qRT-PCR) was used to analyse γ-globin gene expression, and flow cytometry and immunofluorescence microscopy for evaluating HbF production. Cinchona alkaloids showed dose dependent erythroid differentiation, time driven cellular proliferation, with kinetics of hemoglobin accumulation in K562 cells. The findings of qRT-PCR showed an increase in expression of γ-globin mRNA content (3.17-fold in cinchonidine and 2.03-fold increase in quinidine treated K562 cells), accompanied by an increase in fetal hemoglobin production. Altogether, this study demonstrates that cinchona alkaloids can be used as therapeutic agents in treating β-thalassemia after further biological investigation.

Способ получения рекомбинантного гранулоцитарно-макрофагального колониестимулирующего фактора человека

A suggested method for obtaining recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) includes the accumulation of the producer strain biomass enriched with the target product up to 30% of total protein content, its isolation and purification. The later consists of the following stages: ultrasound cell disintegration, washing of inclusion bodies with buffer solutions, GM-CSF solubilization from inclusion bodies by 6 M urea, denaturation-renaturation of protein molecules and purification by chromatography on DEAE-Sepharose and combined chromatography on CM-Sepharose and Q-Sepharose followed by dialysis. The proposed method makes it possible to yield up to 10 mg of the protein preparation from 1 g of wet cells with the purity of 98% and high activity shown on the human erythroleukemia cell line. granulocyte-macrophage colony-stimulating, GM-CSF, producer strain, cultivation, chromatographic purification. The work was performed in the framework of the State Assignment «Adjustment of the Technology of Preparative Obtaining and Purification of Recombinant Proteins» (no. 13/18).

RAB31-Mediated Endosomal Trafficking Is Defective in RUNX1 Haplodeficiency

RAB GTPases are key players in vesicle trafficking, granule targeting of proteins, granule biogenesis and secretion. RAB31 (Ras-related protein 31) has been implicated in the regulation of vesicular trafficking between the Golgi/TGN and endosomes, and from early endosome (EE) to late endosome (LE). Studies in neuronal cells have implicated RAB31 in the transport of epidermal growth factor receptor (EGFR) from EE to LE. RUNX1, a major hematopoietic transcription factor, plays a critical role in megakaryocyte (MK) maturation and platelet production. Patients with RUNX1 haplodeficiency have thrombocytopenia, abnormal platelet function, and impaired granule contents and secretion. Our studies in a patient with a heterozygous RUNX1 mutation (c.969-323G>T ) (Sun et al, Blood 103: 948-54, 2004) associated with thrombocytopenia, platelet dysfunction, granule deficiency and impaired platelet responses revealed decreased platelet expression of RAB31. The role of RAB31 in MK and platelets is unknown. We addressed the hypothesis that RAB31 is a transcriptional target of RUNX1. We studied the role of RAB31 on vesicle transport in MK cells in human erythroleukemia (HEL) cells transformed into megakaryocytic cells by PMA (phorbol 12-myristate 13-acetate). RAB31 mRNA was decreased on platelet expression profiling of the patient (fold change: 0.28, p<0.0076, Sun et al J Thromb Haemost 5: 146-154). With real-time PCR platelet platelet RAB31 mRNA was decreased compared to 5 healthy controls by 60-80% in our patient (P1) and in two additional unrelated patients (siblings, P2 and P3) with RUNX1 mutation (c.508+1G>A). Platelet RAB31 protein was decreased compared to that in 5 healthy controls in patients P2 and P3. RAB31 promoter region (-2023/-1bp from the ATG) revealed 4 RUNX1 consensus sites: site I (-813/-808), site II (-972/-967), site III (-1500/-1495) and site IV (-2007/-2002). Chromatin immunoprecipitation (ChIP) revealed RUNX1 binding to RAB31 sites II and IV but not to other sites. Electrophoretic mobility shift assays using HEL cell proteins showed RUNX1 binding to sites II and IV. In luciferase reporter assays, mutation of individual sites II and IV decreased promoter activity indicating that they are functional sites. RAB31 promoter activity and protein expression were inhibited by RUNX1 siRNA and enhanced by RUNX1 overexpression. These indicate that RAB31 is a direct RUNX1 target, providing a mechanism for decreased RAB31 in patient platelets. We investigated putative roles of RUNX1 and its target RAB31 in endosomal dynamics in PMA treated HEL cells. We used immunofluorescent staining for markers of early endosomes (EE, EEA1) and late endosomes/multivesicular bodies (LE/MVB, CD63) in RUNX1- or RAB31-depleted cells by siRNA transfection. Either RUNX1 or RAB31 siRNA yielded a striking enlargement of early endosomes, as indicated by the EE marker EEA1. This finding suggests a role for RUNX1/RAB31 in EE maturation, either by mediating vesicle fission, or maturation to late endosomes by fusion with other endosomal vesicles. This effect of RUNX1 knockdown on EE enlargement was partially reversed by reconstitution of RAB31 by plasmid co-transfection, indicating that RAB31 is a significant but non-exclusive contributor to this RUNX1 function.Conclusions: These studies provide the first evidence that RAB31 is a direct transcriptional target of RUNX1 and a mechanism for RAB31 downregulation in RUNX1 haplodeficient patients. Downregulation of RAB31 or RUNX1 results in impaired endosomal maturation/trafficking, and this may contribute to the defective handling of α-granule proteins recognized in patients with RUNX1 mutations. DisclosuresLambert:Sysmex: Consultancy; Rigel: Consultancy; Bayer: Membership on an entity's Board of Directors or advisory committees; Educational Concepts in Medicine: Consultancy; CSL: Consultancy; Novartis: Membership on an entity's Board of Directors or advisory committees; Summus: Consultancy; Amgen: Membership on an entity's Board of Directors or advisory committees; Shionogi: Consultancy.