Unit Assay Research Articles

TFF3 promotes pituitary tumor cell migration and angiogenesis via VEGFA.

Pituitary adenoma is a typical adult primary brain tumor and 35% of pituitary adenomas are invasive. The enhancement of angiogenesis is essential for the spread and invasiveness of invasive pituitary adenoma. Thus, it is urgent to uncover the mechanism and relevant biomolecular targets for the therapy and prognosis of pituitary adenomas. The HP75 cells were transfected with si‑NC, si‑TFF3, pcDNA, and pcDNA‑TFF3 to investigate the effects of TFF3 on the proliferation, migration and invasion of pituitary tumor cell. The protein level of TFF3 and HIF‑1α/VEGFA was determined by western blot. The transwell migration assay and wound healing assay were used to investigate the influence of TFF3 on the cell migration and invasion of HP75 cells. The tumor angiogenesis was determined by tube formation assay. The proliferation of HP75 cells was assessed by using MTT assay and colony‑forming unit assay. The cell proliferation rate was separately enhanced and reduced remarkably in TFF3 overexpression group and si‑TFF3 group. TFF3 could modulate the proliferation, migration and invasion ability of HP75 cells. Furthermore, TFF3 may play a oncogenic role in HP75 cells. Overexpression of TFF3 enhanced the number of branching points and network formation in HP75 cells, suggesting the TFF3 had positive effects on cell angiogenesis. These results also disclosed a novel relationship between TFF3 expression and the activation of the HIF‑1α/VEGFA signaling pathway. In summary, this study uncovered new insight into the mechanisms of TFF3's anti‑tumor activities in pituitary adenoma cells by investigating its effects on HIF‑1α/VEGFA signaling pathway regulation.

#40 An RSV live-attenuated vaccine candidate lacking G protein mucin domains is highly attenuated, immunogenic, and effective in RSV prevention in BALB/c mice

Abstract Background RSV is a leading viral respiratory pathogen in infants. Live attenuated vaccines (LAV) show promise to reduce the disease burden of RSV disease. The RSV attachment protein (G protein) is heavily glycosylated with mucin domains that promote viral attachment. They may also aid in immune evasion through steric shielding of immunogenic epitopes. The objective of this study was to create and rescue RSV LAV candidates lacking the RSV G protein mucin domains with attenuated LAV replication and increased immunogenicity. Method LAV vaccine candidates “G155” and “G155-S” were generated from recombinant A2-line19F strain RSV by deletion of the G-protein mucin domains. G155-S additionally underwent deletion of the transmembrane domain, such that G155-S only expressed the secreted form of the G protein, while G155 expressed both transmembrane and secreted forms. Western blotting was used to characterize F and G protein expression in both vaccine candidates in comparison to wild-type (wt) A2-line19F. Enzyme-linked immunosorbent assays (ELISAs) were used to measure monoclonal antibody binding to LAV virus lysate. Viral growth kinetics were measured in HEp2 cells and in BALB/c mouse lungs by a fluorescent focus unit (FFU) assay. Immunogenicity was determined by measuring serum neutralizing antibody titers in BALB/c mice following a prime/boost regimen on days 0 and 28. Mice were challenged with wt RSV on day 60, and LAV efficacy was determined by measuring RSV lung viral loads by FFU assay and RT-PCR on day 4 post-RSV challenge. Results The vaccine candidates showed increased monoclonal antibody binding to surface F protein by ELISA, despite similar levels of F protein expression to wt, consistent with F epitope de-shielding. Both vaccine candidates were significantly attenuated in vitro and were undetectable in mouse lungs via FFU assay. By RT-PCR, LAV viral RNA was reduced 3-fold for G155-S, and 22-fold for G155 compared to wt RSV in mouse lungs. Both vaccine candidates elicited similar neutralizing antibody titers compared to wt A2-line19F on day 28 (G155 GMT 13, 95% CI 3, 53; G155-S GMT 10, 95% CI 2, 51; A2-line19F GMT 13, 95% CI 12, 15), and significant increases in titers were observed on day 59 (G155 GMT 440, 95% CI 5, 36057; G155-S 436, 95% CI 354, 537; A2-line19F 275, 95% CI 134, 567). Following RSV challenge, vaccinated mice showed no detectable lung virus by FFU assay and a reduction in lung viral RNA by RT-PCR of 560-fold for G155 and 604-fold for G155-S compared to RSV-challenged unvaccinated mice. Conclusion G155 and G155-S are highly attenuated RSV LAV candidates that protect against RSV challenge in mice and produce high levels of neutralizing antibodies comparable to wt RSV.

Inosine monophosphate dehydrogenase type1 sustains tumor growth in hepatocellular carcinoma.

BackgroundInosine monophosphate dehydrogenase (IMPDH) is the key enzyme in the biosynthesis of purine nucleotides. IMPDH1 and IMPDH2 are the two isoforms of IMPDH and they share 84% amino acid similarity and virtually indistinguishable catalytic activity. Although high expression of IMPDH2 has been reported in various cancers, the roles of IMPDH1 in hepatocellular carcinoma (HCC) are largely unknown.MethodsThe expression and the clinical relevance of IMPDH1 in 154 HCC patients were detected by immunohistochemistry analysis. The stable IMPDH1 knockdown HuH7 cells were established by lentiviral RNAi approach. The single cell proliferation was detected by colony‐forming unit assay. The tumor initiation and growth ability were measured by using xenograft tumor model in immunodeficient mice. The effect of IMPDH1 on cellular signaling pathways was analyzed by genome‐wide transcriptomic profiling.ResultsThe expression of IMPDH1 is upregulated in tumor tissue compared with adjacent liver tissue, and higher expression of IMPDH1 is associated with better patient cumulative survival. In experimental models, loss of IMPDH1 in HCC cells inhibits the ability of single cell colony formation in vitro, and reduces the efficiency of tumor initiation and growth in immunodeficient mice. Consistently, loss of IMPDH1 results in distinct alterations of signaling pathways revealed by genome‐wide transcriptomic profiling.ConclusionIMPDH1 sustains HCC growth and progression.

Abstract 14015: Extracellular Vesicle-Associated microRNAs From the Plasma of HIV-Positive Individuals Enhance Atherosclerosis Burden

Introduction: People living with HIV (PLHIV) have a higher risk of cardiovascular diseases (CVD) compared to HIV negative (HIV - ) individuals. The molecular mechanisms underlying the increased CVD risk remain elusive. Extracellular vesicles (EVs) mediate cell-to-cell communication through cargo transfer, including microRNAs (EV-miRs). Hypothesis: HIV infection results in modification of plasma EVs cargo and these HIV positive EVs (HIV posEVs ), specifically EV-miRs, recapitulate the effects of HIV infection on CVD development. Methods: Atherosclerosis burden was assessed by carotid ultrasound in 74 PLHIV treated with antiretroviral therapy (ART) with viral loads <50 c/ml and 23 HIV - subjects. Circulating endothelial progenitor cell (EPC) levels were measured by colony forming unit (CFU) assay. PLHIV plasma was fractionated into plasma EVs (HIV posEVs ) and plasma-depleted of EVs (HIV plasmadepEVs ). These fractions, EVs from HIV - subjects (HIV negEVs ) and PBS were injected weekly into apoE -/- mice via tail vein for three months. Atherosclerosis burden (H&E and Oil Red O staining), bone marrow (BM) EPC senescence (β-galactosidase staining), proliferation (MTT assay) and migration (“scratch” assay) were examined. RNASeq was used to identify EV-miRs that mediated the effects of HIV posEVs . Tailored EVs (TEVs) loaded with antagomirs for top EV-miRs were generated to determine their capacity to reverse the adverse effects of HIV posEVs in mouse EPCs in vitro . Results: PLHIV had higher atherosclerosis burden and lower circulating EPC numbers. Mice treated with HIV posEVs showed increased atherosclerotic burden, higher BM EPC senescence and lower functionality. HIV posEVs also negatively affected mouse BM EPC senescence in vitro . RNAseq identified four top candidate EV-miRs upregulated in HIV posEVs : let-7b-5p, miR-16-5p, miR-423-5p, and miR-103a-2. TEVs loaded with the antagomirs for let-7b and miR103a-2 counteracted the effects of HIV posEVs on the accelerated senescence of mouse BM EPCs. Conclusion: 1) Plasma EVs from PLHIV reconstitute the effects of HIV infection on accelerated atherosclerosis via negatively affects EPC functionality; and 2) TEVs, targeting candidate EV-miRs counteracts the effects of HIV infection on EPCs.

Modeling clonal hematopoiesis in umbilical cord blood cells by CRISPR/Cas9

To investigate clonal hematopoiesis associated gene mutations in vitro and to unravel the direct impact on the human stem and progenitor cell (HSPC) compartment, we targeted healthy, young hematopoietic progenitor cells, derived from umbilical cord blood samples, with CRISPR/Cas9 technology. Site-specific mutations were introduced in defined regions of DNMT3A, TET2, and ASXL1 in CD34+ progenitor cells that were subsequently analyzed in short-term as well as long-term in vitro culture assays to assess self-renewal and differentiation capacities. Colony-forming unit (CFU) assays revealed enhanced self-renewal of TET2 mutated (TET2mut) cells, whereas ASXL1mut as well as DNMT3Amut cells did not reveal significant changes in short-term culture. Strikingly, enhanced colony formation could be detected in long-term culture experiments in all mutants, indicating increased self-renewal capacities. While we could also demonstrate preferential clonal expansion of distinct cell clones for all mutants, the clonal composition after long-term culture revealed a mutation-specific impact on HSPCs. Thus, by using primary umbilical cord blood cells, we were able to investigate epigenetic driver mutations without confounding factors like age or a complex mutational landscape, and our findings provide evidence for a direct impact of clonal hematopoiesis-associated mutations on self-renewal and clonal composition of human stem and progenitor cells.

MicroRNA‑502‑3p promotes Mycobacteriumtuberculosis survival in macrophages by modulating the inflammatory response by targeting ROCK1.

Tuberculosis (TB) is caused by Mycobacterium tuberculosis (M. tuberculosis) infection and has the highest mortality rate of any single infectious disease worldwide. The aim of the present study was to investigate the function of microRNA (miR)-502-3p in M. tuberculosis-infected macrophages. The Gene Expression Omnibus database was used to analyze miR-502-3p expression in patients with TB and healthy individuals. THP-1 and RAW 264.7 cells were transfected with miR-502-3p mimic, miR-502-3p inhibitor, pcDNA3.1-ROCK1 or their negative controls. The expression levels of miR-502-3p and inflammatory cytokines were evaluated using reverse transcription-quantitative PCR. The colony-forming unit assay was performed to assess the survival of M. tuberculosis in macrophages, and Toll-like receptor (TLR)4/NF-κB signaling pathway-associated protein expression levels were detected by western blotting. The nuclear translocation of NF-κB p65 was detected via immunocytochemistry. TargetScan was used to predict the binding sites between miR-502-3p and ROCK1. The interaction between miR-502-3p and Rho-associated coiled-coil-forming protein kinase 1 (ROCK1) was confirmed using a dual-luciferase reporter assay; ROCK1 was demonstrated to be a direct target gene of miR-502-3p. Results from the present study demonstrated that miR-502-3p expression was significantly increased during M. tuberculosis infection in macrophages. Upregulation of miR-502-3p expression levels significantly enhanced the survival of intracellular M. tuberculosis. IL-6, TNF-α, and IL-1β mRNA expression levels were significantly upregulated during M. tuberculosis infection but were downregulated by miR-502-3p overexpression. Moreover, miR-502-3p mimics transfection significantly downregulated TLR4/NF-κB signaling pathway-associated protein expression and significantly reduced nuclear transcription of NF-κB in M. tuberculosis-infected macrophages. ROCK1 overexpression reversed the miR-502-3p inhibitory effect on cytokine production in M. tuberculosis-infected macrophages. In conclusion, miR-502-3p/ROCK1 may serve an anti-inflammatory role and may improve the survival of M. tuberculosis within macrophages, which may provide a promising therapeutic target for TB.

Oncogenic SETBP1 Mutations Combine with Activating Mutations in CSF3R to Produce a Highly Proliferative, Lethal Leukemia through Aberrant Myc Signaling

SETBP1 (SET Binding Protein 1) mutations are associated with exceptionally poor prognosis in myeloid neoplasms. Despite this, SETBP1's role in oncogenesis remains incompletely understood. In this study, we find that SETBP1 leads to a marked upregulation of the Myc oncogene and associated pro-stem and progenitor programs through epigenetic dysregulation. We further identify an epigenetic modulatory drug that normalizes SETBP1-driven Myc overexpression and synergizes with disease-relevant therapy. SETBP1 has documented roles in both the regulation of tumor suppressor pathways and modulation of transcription. To understand how SETBP1 modulates leukemia biology and leverage this mechanistic insight to develop novel therapeutic strategies, we turned to a genetically well-defined model system, chronic neutrophilic leukemia (CNL). SETBP1 is mutated in approximately half of all cases of CNL, a myeloproliferative neoplasm characterized by the presence of signaling-activating mutations in Colony Stimulating Factor 3 Receptor (CSF3R). By expressing SETBP1 and CSF3R mutations in mouse hematopoietic progenitors, we have generated models of CNL that can be leveraged for mechanistic studies and drug development. In a hematopoietic colony forming unit (CFU) assay, murine hematopoietic progenitors co-expressing mutant SETBP1 and mutant CSF3R have a high proliferation phenotype compared to those with mutant CSF3R alone. When cells co-expressing mutant SETBP1 and CSF3R are transplanted into lethally irradiated mice, they develop a rapidly lethal disease relative to the control mice. This is associated with an expansion of the granulocyte lineage, quantified by complete blood count, flow cytometry and histology. We find that SETBP1 is essential for the induction of a pro-proliferative transcriptional program. One of the most prominent SETBP1-associated signatures is that of Myc target genes. Myc itself is one of the top differentially expressed genes driven by SETBP1. Congruent with its increased expression, we also find higher Myc transcriptional activity in cells overexpressing SETBP1. To better understand the epigenetic regulation of Myc and progenitor pathways by SETBP1, we employed a low input profiling methods called CUT&Tag to assess the activation of regulatory elements. We began by profiling two epigenetic marks associated with active enhancers-H3K4me1 and H3K27Ac. We also assessed activation of the Myc promoter by measuring H3K27Ac and H3K4me3. Together this data helps us to understand the epigenetic underpinnings for dysregulation of Myc-driven programs by SETBP1. Therapeutic strategies that normalize aberrant Myc activity may be effective against SETBP1-driven disease. We found that SETBP1 and CSF3R transformed hematopoietic progenitors are highly sensitive to inhibitors of the epigenetic regulator lysine specific demethylase 1 (LSD1). LSD1 inhibition restores Myc expression to physiological levels and represses Myc promotor activity in vitro. Furthermore, LSD1 inhibitors are highly synergistic with JAK inhibitors, which block signaling downstream of CSF3R. Together these data establish the importance of Myc activation in SETBP1-driven malignancies and identify a therapeutic approach to normalize aberrant Myc activity. In future, we will use the insight gained in this genetically well defined disease to inform studies on the mechanistic basis and therapeutic vulnerabilities of other SETBP1-mutant myeloid malignancies. Disclosures Maxson: Gilead Sciences: Research Funding; Ionis Pharmaceuticals: Other: Joint oversight committee for a collaboration between OHSU and Ionis Pharmaceuticals.

Astragalus polysaccharide has a protective effect on hematopoiesis in an irradiated mouse model and decreases apoptosis in megakaryocytes.

Huangqi, the dried root of Radix Astragali, is an essential herb in Traditional Chinese Medicine and has been used to promote hematopoiesis for centuries. Astragalus polysaccharide(ASPS), the bioactive compound of Huangqi, serves a crucial role in hematopoiesis. The aim of the present study was to investigate the hematopoietic effects, in particular the thrombopoietic effects, and the molecular mechanisms of ASPS using an irradiation‑induced myelosuppressive mouse model. Colony‑forming unit assays, flow cytometric analysis of apoptosis, ELISAs, Giemsa staining and western blotting were performed to determine the hematopoietic and anti‑apoptotic effects of ASPS. The results demonstrated that ASPS enhanced the recovery of red blood cells at day21 following treatment, as well as platelets and white blood cells at day14. In addition, ASPS promoted colony formation in all lineages (megakaryocytes, granulocyte monocytes, erythroid cells and fibroblasts). The morphological study of the bone marrow demonstrated that tri‑lineage hematopoiesis was preserved in the ASPS‑ and thrombopoietin(TPO)‑treated groups compared with the control group. The overall cellularity (mean total cell count/area) of the ASPS‑treated group was similar to that of the TPO‑treated group. Additionally, <em>invitro</em> experiments indicated that treatment with 100<em>µ</em>g/ml ASPS exhibited the maximum effect on colony formation. ASPS attenuated cell apoptosis in megakaryocytic cells via inhibiting the mitochondrial caspase‑3 signaling pathway. In conclusion, ASPS promoted hematopoiesis in irradiated myelosuppressive mice possibly via enhancing hematopoietic stem/progenitor cell proliferation and inhibiting megakaryocytes apoptosis.

Abstract 487: Safety and functional profiling of clinical grade mesenchymal stem cells derived from bone marrow, adipose tissue and Wharton's jelly for cancer therapy

Abstract Background: Mesenchymal stem cells (MSCs) have the potential as a drug delivery tool in cancer immunotherapy. We previously demonstrated that MSCs can be used to deliver BCG immunotherapy to the hypoxic core of tumors (#AACR-Abstract-3903). However, lack of clinical grade MSCs hamper the use of MSCs in clinical practice. The production of clinical grade human derived MSCs in accordance with current Good Manufacture Practice (cGMP) is an unmet need in the cancer immunotherapy. However, the selection criterion for characterizing the safety of MSC-based products is not well cited yet. In an effort to generate a potent therapeutic cell based product, we design a series of in-vitro safety assays for the production of high quality tissue specific human MSCs (hMSCs) as a final products before releasing from cGMP. Method: Here, we have isolated MSCs from Bone marrow (BM), Adipose tissue (AD) and Wharton Jelly (WJ) and expanded in serum free media. The in-vitro proliferation and phenotypic profiling of stem cell markers performed to check purity of the ex-vivo cultured hMSCs. To determine the clinical safety, we have done stemness marker analysis and cellular senescence capacity. To determine the functional aspects, tri-lineage differentiation and immunopotency of hMSCs were performed. Additionally, to assure the in vitro anti-tumorigenic properties of characterized hMSCs subjected to 2D colony forming unit (CFU) assay, 3D clonogenic assay as 3D organoid culture system and karyotyping. Results: Here, we proposed bioprocess design that allowed successful isolation of MSCs from BM, AD and WJ separately. Growth profiles of all isolated MSCs (n = 3) under serum free condition media with respect to serum containing media shows an exponential growth phase. The decline phase started after Day 7. Results also shows significant variation in population doubling time (PDT) of all isolated hMSCs at Passage 3 (n = 3) and Passage 5((n = 3) in comparison between serum free condition media &amp; serum containing media. We found all hMSCs cultured in serum free media shows positive for CD105-APC (&amp;gt;80%), CD73-PE (&amp;gt;80%), CD90- PerCP (&amp;gt;80%), CD29- FITC (&amp;gt;80%) and HLAI (&amp;gt;80%), and negative HLAII- FITC(&amp;lt;2.20 %) and CD34/45- FITC (&amp;lt;2.20 %) expressions at Passage 3 &amp; passage 5, indicating a stem cell phenotype (n = 3). Isolated hMSCs showed osteocyte, adipocytes and chondrocytes differentiation and high capacity to inhibit the proliferation of activated lymphocytes. Additionally, hMSCs positively expressed Nanog and Sox-2 stemness gene and cellular senescence from passage 7. In 2D CFU assay these cells showed colony forming capacity and in 3D organoid culture system in soft agar based matrix, we found that these cells are non-tumorigenic and didn't showed any karyotypic alteration. Conclusion: The collective result provides compelling evidence of the clinical safety of cGMP-compliant BM-MSCs, AD-MSCs and WJ-MSCs potentially suitable for future therapeutic application. Citation Format: Sorra Sandhya, Sonali Rawat, Suchi Gupta, Harshita Sharma, Bikul Das, Sujata Mohanty. Safety and functional profiling of clinical grade mesenchymal stem cells derived from bone marrow, adipose tissue and Wharton's jelly for cancer therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 487.

Preeclampsia in pregnancy affecting the stemness and differentiation potency of haematopoietic stem cell of the umbilical cord blood

BackgroundUmbilical cord blood (UCB) has been proposed as the potential source of haematopoietic stem cells (HSC) for allogeneic transplantation. However, few studies have shown that a common disease in pregnancy such as preeclampsia would affect the quality of UCB-HSC. Total nucleated cell count (TNC) is an important parameter that can be used to predict engraftment including UCB banking. Colony forming unit (CFU) assay is widely used as an indicator to predict the success of engraftment, since direct quantitative assay for HSC proliferation is unavailable. The aim of this study is to investigate the effects of preeclampsia in pregnancy on the stemness and differentiation potency of UCB-HSC.MethodsMononuclear cells (MNC) were isolated from UCB and further enriched for CD34+ cells using immune-magnetic method followed by CFU assay. A panel of HSC markers including differentiated haematopoietic markers were used to confirm the differentiation ability of UCB-HSC by flow cytometry analysis.Results/ discussionThe HSC progenitor’s colonies from the preeclampsia group were significantly lower compared to the control. This correlates with the low UCB volume, TNC and CD34+ cells count. In addition, the UCB-enriched CD34+ population were lymphoid progenitors and capable to differentiate into natural killer cells and T-lymphocytes.ConclusionThese findings should be taken into consideration when selecting UCB from preeclamptic mothers for banking and predicting successful treatment related to UCB transplant.

Atomic oxygen radical‐induced intracellular oxidization of mould spore cells

AbstractThe inactivation mechanism of mould spores using plasma‐generated neutral reactive oxygen species (ROS) was investigated in this paper. Typical cell viability using a counting of colony‐forming unit assay indicated a major state of the spore cells. Both intracellular and extracellular damages have been investigated by a couple of well‐established cell visualization techniques: confocal fluorescence emission microscopy, scanning electron microscopy, and transmission electron microscopy. It is revealed in this paper that the combination of the spore cell viability with those visualization results strongly suggested an early stage of the cell inactivation when ROS can induce intracellular lipid peroxidation through both nanometer‐thick lipid cell membrane and less‐damaged several hundred nanometer‐thick cell wall.

Active DNA Demethylation Mediated By GADD45β Is Essential during G-CSF Triggered Granulocytic Differentiation

Congenital neutropenia (CN) is a bone marrow failure syndrome which is characterized by a maturation arrest of myeloid progenitors in the bone marrow. The pathomechanism leading to the defective granulopoiesis in CN patients is not fully elucidated. We recently reported the inability of G-CSF to induce GADD45β (Growth arrest and DNA damage-inducible 45, beta) expression in myeloid progenitors of CN patients harboring ELANE or HAX1 mutations. GADD45β functions as stress sensor downstream of myeloid cytokines and is important in murine stress-induced hematopoiesis. Moreover, we could show that C/EBPα, which is severely diminished in CN cells, binds and activates the GADD45β promoter. Importantly, rescue of the GADD45β expression in ELANE-CN HSPCs by lentivirus-based transduction of GADD45B cDNA restored defective granulocytic differentiation compared to control transduced cells. Transduction of mouse HSCs with ectopic Gadd45β resulted in drastic decrease of immature and increase of mature myeloid cells over time. Colony forming unit (CFU) and CFU re-plating assay showed that Gadd45β overexpressing mouse HSCs exhibit lower proliferation capacity. To determine the role of GADD45β in vivo, we performed CRISPR/Cas9 knockout of gadd45bb in wild type and Tg(mpo:gfp) zebrafish embryos, in which GFP expression is activated by the mpo promoter. We found that gadd45bb-deficiency lead to dramatic reduction in neutrophil numbers as assessed by counting mpo+ cells. Ectopic expression of G-CSF using a heat-inducible system increased the number of mpo+ cells in gadd45bb crispants but not to a level similar to wild type zebrafish expressing G-CSF. GADD45 proteins are implicated in active DNA demethylation by recruiting the DNA repair machinery to specific sites in the genome to convert 5-methylcytosine into unmethylated cytosine. Nothing is known about active DNA demethylation downstream of G-CSF and the role of GADD45β in this process. We sought to unveil whether GADD45β mediates G-CSF triggered myeloid differentiation by specific gene demethylation. To address this, we performed RNA sequencing and DNA methylation analysis of wild type (WT) and GADD45β knockout (KO) CD34+ HSPCs treated or untreated with G-CSF for 72h. The RNA-seq analysis demonstrated a GADD45β-dependent upregulation of genes which play a role in neutrophil differentiation and function as for instance RXRA, ITGAM (CD11b), FPR1/2, MEFV and BATF upon G-CSF treatment. Strikingly, these genes were also upregulated in promyelocytes of healthy controls upon G-CSF treatment, but not in CN patient promyelocytes. Genomatix-based gene ontology analysis of significantly upregulated genes in the G-CSF-treated WT but not GADD45β KO group displayed that diseases associated with this expression signature included neutropenia, leukocyte disorders and AML. Motif enrichment analysis of GADD45β-responsive genes predicted mainly motifs for myeloid-specific transcription factors (e.g. RXRA, SPI1 and C/EBPβ) to be enriched. Finally, DNA methylation analysis revealed significant differences in methylation pattern between GADD45β WT and KO groups upon G-CSF treatment. In WT cells G-CSF caused the hypomethylation of 13.516 CpGs and in GADD45β KO cells, only 8.440 CpGs were hypomethylated. Methylation changes between WT and KO groups mainly occurred in myeloid-specific genes, e.g. ELANE, MPO, CSF3R and AZU1. We also identified myeloid differentiation-related genes of which mRNA expression was regulated by GADD45β-mediated DNA demethylation, as for instance RXRA, CXCR1, MEFV, FPR2 and SERPINA1. Importantly, diseases associated with the defective gene demethylation downstream of G-CSF were neutropenia, immune system diseases, pre-leukemia and AML, as assessed by Genomatix analysis. Taken together, we report for the first time the importance of GADD45β-dependent DNA demethylation for human and zebrafish G-CSF-triggered neutrophil differentiation. The failure of G-CSF to induce GADD45β in CN patients might be a reason for the maturation arrest of granulopoiesis. Disclosures No relevant conflicts of interest to declare.

Loss of METTL3 Mediated m6A RNA Modification Results in Double-Stranded RNA Induced Innate Immune Response and Hematopoietic Failure

Hematopoietic stem cell (HSC) self-renewal and lineage output are orchestrated by multiple regulatory layers, including RNA modifications. N6-methyladenosine (m6A) is an abundant modification found in RNAs which affects the translation and stability of modified transcripts. The effects of m6A are determined by m6A writers (install m6A), erasers (remove m6A) and readers (recognize m6A). In embryonic stem cells, deletion of the m6A writer METTL3 enforces a naïve pluripotent state. This raises the question of whether m6A RNA methylation analogously regulates stem cell self-renewal and differentiation in somatic stem cells such as HSCs. Using a Vav-Cre/Mettl3 (VCM3) hematopoietic-specific knockout mouse model, we show that loss of the RNA m6A writer METTL3 in fetal HSCs results in hematopoietic failure and perinatal lethality. At E14.5 (FL) hematopoiesis loss of Mettl3/m6A results in hematopoietic failure with expansion of Lin-Sca-1+c-Kit+ (LSK) hematopoietic stem and progenitor cells (HSPCs) that are defective in the production of progenitors and mature blood cells, as evidenced by failure to rescue lethally irradiated congenic recipient mice in transplant experiments. The relative defect I hematopoiesis was further demonstrated by competitive transplant experiments, in which transplanted KO FL were consistently out-competed by WT FL. Interestingly, BrdU/7AAD labeling reveals a significant proliferative defect with reduced BrdU uptake in VCM3 KO FL cells, and specifically in Lin-c-Kit+Sca-1- (LK) progenitor cells. RNA-seq analysis of FL LSK cells reveals that loss of m6A results in upregulation of multiple 2'-5'-oligoadenylate synthetase (OAS) family genes. Interestingly, the majority of OAS family genes are not m6A modified in several m6A sequencing data sets. We therefore hypothesized that the OAS genes might be regulated at the transcriptional level. We performed cleavage under targets and release using nuclease (CUT&amp;RUN) analysis and found that OAS family genes are transcriptionally activated, as evidenced by significant increase in H3K4 trimethylation (H3K4me3) at their respective promoter regions. The OAS family genes are activated by the presence of double stranded RNA (dsRNA), which can arise either endogenously or as a pathogen-associated trigger of the innate immune system in the context of viral infection. The dsRNA response includes three major response mechanisms. First, OAS genes facilitate RNase L dimerization, which mediates cleavage of cellular tRNA and rRNA, resulting in translation and proliferation arrest. Second, activation of the PKR/eIF2a pathway also results in translation arrest. Lastly, activation of the MDA-5/RIG-I/MAVS response induces interferon signaling, which further limits cellular proliferation. Staining of VCM3 WT &amp; KO FL cells with the dsRNA-specific J2 antibody directly demonstrated significant accumulation of dsRNA in KO FL cells. We further demonstrated activation of the OAS/RNaseL axis in KO cells by quantification of tRNA-His-36 cleavage, which acts as a sensitive marker of RNase L activity. Phosphorylation of both PKR and eIF2a in KO cells demonstrated enhanced activity of this pathway. Lastly, we found a significant upregulation of interferon pathway genes in KO cells, and demonstrated that CRISPR mediated knockout of Mavs in VCM3 KO FL cells diminishes the activity of interferon response genes and rescues the Mettl3 KO phenotype in colony forming unit (CFU) assays. These results suggest that in the absence of m6A, endogenous dsRNA formation is enhanced, resulting in an associated inflammatory response which partially accounts for the hematopoietic defect observed in Mettl3 KO mice. In conclusion, our study suggests a novel, protective role for the m6A RNA modification in preventing endogenous dsRNA formation and aberrant activation of a detrimental innate immune response during mammalian hematopoietic development. Disclosures Fan: IsoPlexis: Other: served on the scientific discovery board, Patents &amp; Royalties; Singleron Biotechnologies: Other: served on the Scientific Advisory Board; BioTechne: Other: served on the Scientific Advisory Board. Flavell:Rheos Biomedicines: Equity Ownership; GSK: Consultancy; Artizan Biosciences: Equity Ownership; Troy: Equity Ownership; SMOC: Equity Ownership; Zai labs: Consultancy.

Functional Impact of MacroH2A Histone Variants in Acute Myeloid Leukemia

Histone variants are emerging as key regulatory molecules in cancer. Recent data provided first evidence of how transcriptional deregulation and changes in the deposition of histone variants may affect malignant transformation. H2A variants are highly conserved and consist of 8 members. Among all histone variants, macroH2A variants hold a unique C-terminal macro domain, which is approximately twice the size of its H2A-like histone domain. In mammals, there are 3 macroH2A variants encoded by H2AFY (macroH2A1.1, macroH2A1.2) and H2AFY2 (macroH2A2). MacroH2A variants occupy large repressive domains throughout the genome, exerting a repressive role on transcription. So far, the function of macroH2A proteins in hematopoiesis and leukemic transformation is incompletely understood. Here, we report on a functional role of macroH2A histone variants that may influence cell competition in leukemia and normal hematopoietic stem and progenitor cell (HSPC) function. In published gene expression datasets, macroH2A variants H2AFY and H2AFY2 are highly expressed in hematopoietic stem cells (HSCs) compared to mononuclear cells (both p&lt;0.0001****). Moreover, expression of H2AFY and H2AFY2 appeared even higher in acute myeloid leukemia (AML) (p=0.0750 and 0.0037**, respectively) when compared to the expression level in HSCs. This high expression is independent of different genetic subtypes and AML risk groups. To assess for the functional dependency of leukemic cells on the expression of macroH2A variants, we performed an in vitro CRISPR-Cas9 dropout screen focusing on macroH2A variants in MOLM-13 cells. Genetic inactivation of H2AFY but not H2AFY2 resulted in outcompetition of infected cells against non-infected competitors. This finding could be recapitulated in 3 additional AML cell lines (OCI-AML3, THP-1, HL-60), indicating a broader dependency on H2AFY irrespective of the oncogenic background or underlying driver mutation. To validate the functional impact of H2AFY on leukemia development in vivo, we generated a novel conditional knockout mouse model for H2afy and induced leukemia through retroviral infection of sorted Lineage-Sca1+cKit+ (LSK) cells with the oncogene MLL-AF9. Results of these functional studies will be presented. To understand the functional impact of macroH2A variants on normal HSPC function in vivo, we performed competitive repopulation studies. We used conventional knockout mouse models for the 2 H2afy isoforms: macroH2A1.1 and macroH2A1.2 In addition, for inactivation of both isoforms, we used the conditional macroH2A knockout mouse model. H2afy2 was either inactivated by RNAi or conditionally knockout through Mx1-Cre recombinase activation. In brief, whole bone marrow cells (WBMCs) were transplanted into primary recipient mice in a 1:1 competitive manner and compared to the respective wildtype littermate controls. Peripheral blood (PB) chimerism was monitored on a monthly basis 4 weeks post-transplantation for a total of 16 weeks. We observed no significant difference in the recipients of macroH2A1.1 (n=5/5) or macroH2A1.2 (n=10/9), suggesting both macroH2A isoforms are dispensable for intrinsic functions of HSCs. Remarkably, we found macroH2A2-deficient cells (n=6) competed significantly worse than macroH2A2+/+ (n=5) controls in PB (p=0.0043**) and in WBM (p=0.0043**), indicating a requirement for macroH2A2 in maintenance of HSPCs. These findings could be recapitulated by RNA-mediated inactivation of H2afy2 in sorted HSCs. We observed significant reduction of macroH2A2-depleted hematopoiesis after 16 weeks in WBM (p=0.0286*) and PB (p=0.0079**). Importantly, this functional impairment was less pronounced when investigating multipotent progenitor and committed progenitor cell function in spleen colony-forming unit (CFU) assay in vivo or CFU assay in vitro. Taken together, our data indicate distinct requirements for macrohistone variants in leukemic and normal hematopoietic cells. Dependency of leukemic cells but not normal HSPCs on macroH2A may indicate a potential therapeutic index. Disclosures No relevant conflicts of interest to declare.

P5384Postnatal mouse aorta contains yolk sac-derived haemangioblasts with myeloid and endothelial plasticity and vasculogenic capacity

Abstract Background Macrophages and endothelial cells share an intimate relationship during neovessel formation in different pathophysiological conditions. Recent studies have determined that in some tissues, both cell types are derived embryonically from yolk sac (YS) progenitor cells and are maintained postnatally without contribution from circulating sources. The mechanism by which this local “self-maintenance” occurs is unknown. Purpose We previously identified that mouse arteries contain macrophage and endothelial progenitor cells in their adventitial Sca-1+CD45+ compartment. Here we investigated at a clonal level for the existence of postnatal adventitial haemangioblasts and studied their developmental origins. Methods and results Single cell digests were prepared from murine aortas to perform colony-forming unit (CFU) assays in methylcellulose. Aortic cells from C57BL/6J mice selectively generated macrophage colonies (CFU-M) which contained progenitor cells that displayed &gt;95% positive for expression of CD45, Sca-1, c-Kit, CX3CR1 and CSF1R, but negative for Lineage markers, as well as mature monocyte/macrophage (CD11b, F4/80) and endothelial (CD144) markers. Secondary replating of CFU-M progenitors from adult aortas revealed their self-renewal capacity, with 1 in 10 cells forming new CFU-M. Lineage mapping using Flt3CrexRosamT/mG mice demonstrated that aortic CFU-M progenitors were FLT3-ve, indicating that they were not derived from definitive bone marrow haematopoiesis. CFU-M prevalence in C57BL/6J aortas was highest in neonatal mice and diminished progressively with increasing age (∼100 per 105 cells at P1, ∼15 at 12w, ∼5 at 52w, P&lt;0.01, n&gt;4/gp), consistent with prenatal seeding. Embryonic profiling determined that CFU-M progenitors first appeared in extra-embryonic yolk sac around E9.5 and in aorta-gonad-mesonephros at E10.5, before the emergence of definitive haematopoietic stem cells. Inducible fate-mapping then confirmed that aortic CFU-M progenitors originated from CX3CR1+ and CSF1R+ cells in E9.5 yolk sac. Both yolk sac and postnatal aortic CFU-M progenitors generated vascular-like networks when cultured in Matrigel in vitro, containing M2-like macrophages (CD11b+F4/80+CD206+) and endothelial cells (CD31+CD144+). They produced similar progeny and rescued adventitial vascular sprouting when seeded around aortic rings whose adventitia had been stripped. Finally, adoptive transfer of CFU-M progenitors into a mouse model of hindlimb ischaemia resulted in 80% augmentation in hindlimb perfusion compared to cell-free control, with de novo transformation of donor cells into macrophages, endothelial cells and perfused neovessels (n=6). Conclusion To the best of our knowledge, this is the first ever definitive proof at a clonal level for the existence of haemangioblasts in postnatal tissue. Adventitial haemangioblasts originate from extra-embryonic YS and are a source of vasculogenesis in the arterial wall, relevant to vasa vasorum formation. Acknowledgement/Funding NHMRC of Australia (GNT1086796, CDF1161506), NHFA (FLF100412, FLF102056) Royal Australasian College of Physicians

P3495Long-term engraftment of human CD271-positive adipose-derived stem cells with pericytic and less-aged gene profile in a mouse model of hindlimb ischemia

Abstract Background Therapeutic angiogenesis using adipose-derived stem cells (ADSCs) is an attractive strategy for ischemic cardiovascular diseases. We previously reported that human CD271+ population of adipose-derived stem cells (ADSCs) promoted neovascularization with enhanced engraftment in a mouse model of hindlimb ischemia. However, whether and how CD271+ ADSCs promote the long-term engraftment is still uncertain. Purpose We aimed to examine whether the angiogenic effect and cell engraftment capacity of CD271+ ADSCs would be sustained in long-term period. Then, comparative gene profiling between CD271+ and CD271- ADSCs were analyzed. Finally, cell proliferation and endothelial differentiation assays were conducted. Methods ADSCs were isolated from subcutaneous adipose tissue of 5 patients received cardiovascular surgery. CD271+ and CD271- ADSCs were sorted from CD45-CD31-CD34+ ADSCs fraction by FACS sorting (Fig. A). Cultured CD271+ and CD271- ADSCs at passage 6 were labeled by PKH26 cell linker dye and used for xenograft experiments. Briefly, athymic nude mice were subjected to hindlimb ischemia and one million of human ADSCs were injected into the ischemic muscles. In control group, PBS was solely injected. At 2 and 5 weeks, neovascularization was evaluated by immunohistochemistry (capillary density using lectin perfusion). Cell engraftment was assessed by counting PKH26-positive cells. Furthermore, we compared gene profiling between CD271+ and CD271- ADSCs by microarray. Proliferative capacity was evaluated by colony-forming unit (CFU) assay with Giemsa staining. In endothelial differentiation assay, CD271+ and CD271- ADSCs were cultured in differentiation induction medium containing vascular endothelial growth factor for 2 weeks and stained with anti-human CD31 antibody. Results Cell therapy using CD271+ ADSCs demonstrated approximately 3-fold more enhanced neovascularization than those using CD271- ADSCs or PBS in histological analysis of capillary density at 2 weeks from cell therapy (Fig. B and C). At 5 weeks, mice treated with CD271+ ADSCs were significantly rescued from limb ischemia and this was accompanied by sustained engraftment of ADSCs (Fig. D). In microarray analysis, the differentially expressed 2167 genes were extracted to classify CD271+ and CD271- ADSCs. Pathway analysis demonstrated CD271 expression on ADSCs was associated with the pathways related to stemness and cell differentiation. Indeed, we found that genes related to cell proliferation (PI3K, Cyclin D, and Cyclin D2) were up-regulated in CD271+ ADSCs. Additionally, we found the pericytic marker nestin which was significantly up-regulated in CD271+ ADSCs. Consistent with these findings, CD271+ ADSCs were more proliferative and capable for endothelial differentiation while CD271- ADSCs were not. FACS and cell therapy experiments Conclusion These results suggest that CD271+ ADSCs possess long-term engraftment and angiogenic capacity due to their less-aged and more pericytic gene profile. Acknowledgement/Funding Japan Society for the Promotion of Science (JSPS) KAKENHI (Tokyo, Japan) Grant Number JP16H06828

The In-vitro Antiviral Activity of Lactobacillus casei MCJ Protein-based Metabolites on Bovine Viral Diarrhea Virus

Bovine viral diarrhea virus (BVDV) is a ubiquitous immunosuppressive etiological agent that is economically important for a wide host range in the livestock industry. Lactobacillus spp. has widely been using in the field of management and treatment of gastro-enteric disease for both human and animal. The ability of Lactobacillus casei MCJ (L. casei) protein-based metabolized (LPM) to suppress BVDV infection in Madin-Darby Bovine Kidney (MDBK) cell line was demonstrated in this study. The protein-based metabolites were extracted from the cultured L. casei to obtain the safest and beneficial form of the probiotic bacteria. The results of cytotoxicity test reveal that LPM has no cytotoxic effect and the cell viability remains more than 80% even after the cells are treated with 3000 μg/mL of LPM. In comparing positive control, 3000 μg/mL concentration of LPM reduced plaque formation by 73.45% whereas 1000, 100 and 10 μg/mL reduced 65.95%, 39.67% and 4.02% consecutively. The results of plaque formation unit (pfu) assay showed that LPM can reduce the viral infection rate. To know the mechanism of LPM for anti-BVDV activity, MDBK cells were exposed to LPM before, after and co-incubation of virus infection. The co-treatment of LPM with BVDV revealed the best results for the decreased viral protein expression by Western blot, RNA copy number by Quantitative real-time PCR and fewer BVDV infected cells by Immunofluorescences assay. The results suggest that the LPM has a potential anti-BVDV activity which could be a prospective application for the prevention and control of BVDV infection in animal.

Intracellular Assays to Monitor Survival and Growth of Yersinia pestis Within Macrophages.

Yersinia pestis is able to survive and replicate within macrophages, while also being able to live in the extracellular milieu of the host. Assays that facilitate better understanding of how Y. pestis survives intracellularly and subverts normal host antimicrobial defenses require the ability to monitor intracellular Y. pestis survival and replication. In this chapter three different assays for monitoring intracellular survival and replication will be described, along with the formulas and methods to quantify and present the acquired data. These assays are fundamental to answering a multitude of questions pertaining to which bacterial factors are important for intracellular survival. Additionally, these assays can be used, with modifications, for other intracellular pathogens of interest. The first assay discussed will be the conventional bacterial enumeration assay, which quantifies bacterial numbers directly through a classic colony forming units (CFU) assay. Quantifying bacterial burden through CFU determination allows for differentiation between intracellular/cell-associated bacteria and extracellular bacteria. However, CFU determination is laborious, does not allow for direct kinetic monitoring of bacterial growth, and is difficult to adapt to high throughput assays. Bioluminescence bioreporters that use luciferase to monitor bacterial numbers allow for simple, plate reader-based, real-time kinetic monitoring of bacterial growth that is amendable to high throughput techniques. Finally, we will describe live cell microscopy using fluorescent bioreporters, which allows for monitoring of bacterial replication in individual cells and the possibility to visualize interactions between bacterial and host proteins during intracellular infection.

Abstract 4809: Bardoxolone-methyl suppresses both androgen receptor and its splice-variant ARv7 in prostate cancer cells to enhance the anti-cancer efficacy of enzalutamide

Abstract Prostate cancer (PCa) is a leading cause of morbidity and mortality in elderly men in the United States. Despite androgen deprivation therapy (ADT), the recurrence of castration resistant prostate cancer (CRPC) remains a significant challenge. Augmented signaling via the full-length androgen receptor (AR) and the constitutively active AR splice variants, especially ARv7, is associated with ADT-resistance. Therefore, a proper understanding of augmented AR signaling and adjunct strategies to suppress both AR and ARv7 levels are critically needed. ADT-induced oxidative stress (Ox-stress) can facilitate CRPC outgrowth. We previously documented that the Ox-stress induced transcription factor, Nrf2 can suppress AR expression and AR-transactivation function in PCa cells. Recently, we also showed that the Nrf2-inducing natural compound, sulforaphane (SFN) can decrease both AR and ARv7 levels in PCa cells, and co-exposure to SFN enhanced the efficacy of anti-androgens. In this study, we examined whether a synthetic triterpenoid drug, Bardoxolone-methyl (CDDO-me), a potent enhancer of Nrf2, can similarly decrease AR and ARv7 expression. Exposure to nanomolar (nM) concentrations of CDDO-me rapidly downregulated AR levels in the androgen-dependent LNCaP cells and androgen-independent C4-2B cells. Exposure to CDDO-me also suppressed both AR and ARv7 levels in the CRPC line, CWR22Rv1. A biphasic effect of CDDO-me on reactive oxygen species (ROS) levels was observed. Pre-exposure to the antioxidant, n-acetyl cysteine (NAC) blocked the AR-suppressive effect of CDDO-me. Molecular mechanistic studies using actinomycin-D (transcription inhibitor), cycloheximide (translation inhibitor), and MG132 (proteasomal inhibitor) suggested that CDDO-me may decrease AR gene expression and increase degradation of AR and ARv7 proteins. Furthermore, exposure to CDDO-me increased proteasomal activity in PCa cells and suppressed the levels of several AR splicing factors, i.e. the heteronuclear ribonucleoproteins A1 and H1 (hnRNP-A1 and hnRNP-H1). Co-exposure to CDDO-me increased the anti-cancer efficacy of enzalutamide (ENZ) as evident by decreased cell-viability (MTT), migration (scratch-wound) and colony forming unit (CFU) assays. Therefore, adjunct treatment with low-dose CDDO-me may be an effective strategy to suppress AR and ARv7 levels and augment the efficacy of anti-androgens. Most importantly, since CDDO-me is in late-stage clinical trials for chronic kidney disease (CKD), its repositioning as a potent AR suppressive agent may be of significant translational value in PCa patients undergoing ADT. Citation Format: Namrata Khurana, Partha Chandra, Hogyoung Kim, Asim B Abdel-Mageed, Suresh Sikka, Debasis Mondal. Bardoxolone-methyl suppresses both androgen receptor and its splice-variant ARv7 in prostate cancer cells to enhance the anti-cancer efficacy of enzalutamide [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4809.

EVALUATION OF ANTIOXIDANT ACTIVITY OF MAGNOLOL IN SACCHAROMYCES CEREVISIAE

Objective: To investigate the antioxidant activity of magnolol in Saccharomyces cerevisiae (S. cerevisiae) model.Methods: Antioxidant activity of magnolol was demonstrated in S. cerevisiae using spot assay, colony forming unit (CFU) assay, detection of reactive oxygen species (ROS) by fluorescent microscopy and spectrofluorometer, lipid peroxidation (LPO) and catalase (CAT) assay.Results: A dose-dependent increase in sensitivity of S. cerevisiae was observed with increase in hydrogen peroxide (H2O2) concentration. At 1.5 mmol concentration of H2O2, we observed 50 % of cell survivability in CFU and spot assay results. The sensitivity of S. cerevisiae to H2O2 was protected by magnolol treatment. In spot assay, magnolol pre-treatment showed the similar growth pattern as that of control and in CFU assay 75 % of survivability was observed. Fluorescence microscopic images and fluorescence intensity levels using 2´, 7´-dichlorodihydrofluoresceindiacetate (H2DCFDA) showed less number of fluorescence cells and 1.2 fold decrease in fluorescence intensity in magnolol pre-treated cells. The cellular protection in the cells pre–treated with magnolol followed by H2O2 was correlated with the decreased LPO and increase in CAT activity was recorded.Conclusion: The result of the present study demonstrates that magnolol protects S. cerevisiae cells from H2O2 induced oxidant mediated cell death.