Induction of Micro RNA-143 and 145 In Pre-Treatment CD34+ Cells From Patients with Myelodysplastic Syndrome (MDS) After In Vitro exposure to Lenalidomide Correlates with Clinical Response In Patients Harboring the Del5q Abnormality

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 123-123 ◽  
Author(s):  
Christopher P. Venner ◽  
Alan F. List ◽  
Thomas J. Nevill ◽  
H. Joachim Deeg ◽  
Gisela Caceres ◽  
...  
Keyword(s):  
Clinical Response ◽  
Mirna Expression ◽  
Fold Change ◽  
Response To Therapy ◽  
Disease Phenotype ◽  
Chromosome 5 ◽  
Cd34 Cells ◽  
Significant Change ◽  
Pre Treatment

Abstract Abstract 123 Background: Lenalidomide is an effective agent in the treatment of MDS particularily in those possesing the deletion 5q (del5q) abnormality. The disease phenotype is partly related to loss of genetic material found in the commonly deleted region (CDR) on the long arm of chromosome 5. Recently implicated genes include miRNA-143, 145 and 146 all of which appear to play a role in innate immune signalling. In this study we examined whether upregulation of miRNAs in pretreatment CD34+ marrow cells from patients with MDS (both del5q and non-del5q) exposed to lenalidomide in vitro predicts for clinical response to the drug. Patients and Methods: In total 31 pre-treatment patient samples were obtained from three North American centres. 11 patients' samples were positive for del5q. All patients had received lenalidomide as per local protocols. Clinical response was defined as per the International Working Group 2000 criteria. Clinical data were available for 26 patients. Hematologic improvement (HI), specifically the eythroid response (HI-E), was examined as the main clinical endpoint. Changes in miR-143, miR-145, miR-146a and miR-146b expression in CD34+ cells after in vitro lenalidomide exposure (10uM for 48 hours) were examined. The CD34- fraction was also analyzed. dCT values were normalized to 5S. A ≥ 1.5 fold change in miRNA expression was considered significant. Results: For the entire cohort mean fold changes in miRNA-143 and 145 post lenalidomide exposure were 1.6 and 1.7, respectively. Little change was seen in miR-146a and miR-146b expression (mean fold change 1.1 and 1.2, respectively). No significant increase was seen in any of the examined miRNA in the CD34 - fraction (Figure 1a). When separated based on del5q status both del5q and non-del5q groups showed a > 1.5 fold increase in expression of miR-143 and miR-145. No significant change in either miR-146a or miR-146b expression was seen (Figure 1b). In the CD34- fraction no significant change was seen in any of the examined miRNAs, irrespective of del5q status (Figure 1c). Examining HI-E, major responses (MR) were seen in 42% of patients (87.5% in the the del 5q cohort (7/8 had a MR) and 22.2% for the non-del5q cohort (4/18 had a MR)). Correlation with clinical response and miRNA expression in the CD34+ fraction is shown in Table I. In the del5q cohort MR was associated with an increase in both miR-143 and miR-145. No correlation with miRNA expression and clinical response was seen in the non del5q cohort. Conclusion: Exposure of CD34+ cells from patients with MDS to lenalidomide results in up regulation of miR-143 and miR-145, irrespective of del5q status. Clinically, upregulation of miR-143 and miR-145 correlates with a HI-E in patients with del5q and may be predictive of response to therapy. Interestingly, the non-responder in the del5q group failed to upregulate miR-145. A similar gene induction pattern was seen in the non-del5q cohort, yet no correlation was seen with clinical outcome. Given that non-del5q patients do not harbor any abnormalities within the CDR of chromosome 5 these miRNAs are unlikely to play a role in their disease phenotype, nor in their response to lenalidomide. Disclosures: Nevill: Celgene: Honoraria. Karsan:Celgene: Research Funding.

Specific MicroRNA Deregulated in Myleoproliferative Neoplasm Are Regulated by JAK2V617F and May Contribute to Aberrant Hematopoiesis.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 965-965
Author(s):  
Xiaoqing Lin ◽  
Monica Buzzai ◽  
Martin Carroll ◽  
Elizabeth Hexner ◽  
Fabricio F Costa ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Mirna Expression ◽  
Colony Formation ◽  
Lentiviral Vector ◽  
Myeloproliferative Neoplasms ◽  
Rna Extraction ◽  
Differentially Expressed ◽  
Loss Of Function ◽  
Cd34 Cells

Abstract Abstract 965 The myeloproliferative neoplasms (MPN), PV, ET and IMF, harbor the same gain-of-function mutation JAK2V617F at a high frequency (∼100%, 70% and 50% respectively). Accumulating evidence suggest that JAK2V617F may not be the initiating event in MPN, and other genetic anomalies play an important role in MPN pathogenesis. We hypothesized that miRNA deregulation contributes to the development of MPN. To test this idea, miRNA expression in CD34+ cells isolated from 8 patient samples (4 PV with JAK2V617F, 3 ET with wild-type JAK2 and 1 IMF with unknown JAK2 status) and 4 healthy controls was determined using a Taqman Low Density Array (TLDA) representing 667 known miRNAs. PV (JAK2V617F) and ET cases (JAK2WT) showed 14 and 78 differentially expressed miRNAs, respectively, when compared to controls. 6 miRNAs were commonly deregulated in PV and ET, while the majority were unique to each disease type. When all MPN patients were grouped and compared to controls, 28 miRNAs were significantly deregulated (p<0.05). These miRNAs differ from those previously reported to be differentially expressed in the peripheral blood of PV patients. Among these 28, mir-214 was down-regulated and mir-410, mir-22* and mir-505* were up-regulated most consistently. Several miRNAs, including mir-135b, mir-542-5p, mir-149, mir-133b and mir-134 were undetectable in normal CD34+ cells and activated in MPN patients. We further hypothesized that some miRNAs are regulated through the action of the mutant JAK2V617F kinase. To test this, miRNA levels were assessed by TaqMan array in HEL and UKE-1 cells (harboring JAK2V617F) treated with 2 μM JAK inhibitor I (Calbiochem) for 20h before RNA extraction. In parallel, miRNA expression as determined by TLDA in TF-1 cells rendered cytokine independent by stable expression of JAK2V617F was compared to that of control TF-1 cells, both cultured overnight in the absence of cytokines. A total of 24 miRNAs were significantly deregulated (>2 fold) in at least two cell line systems. To test which deregulated miRNAs in MPN patients were JAK2 responsive, JAK2 activity was manipulated in HEL and TF-1 cells as described above, and the expression of miRNAs was determined by individual Taqman miRNA assays. mir-1, mir-200a, mir-9, mir-133b, mir-22* and mir-155 were responsive to manipulation of JAK2 activity. miR-155 expression was repressed 50% with the inhibition of JAK2 in HEL cells and stimulated almost 2 fold with the overexpression of JAK2V617F in TF-1 cells. By contrast, mir-214 (downregulated in MPN) and mir-134 (upregulated in MPN) were not responsive to manipulation of JAK2V617F activity in either the gain or loss-of-function systems. To further confirm the ability of JAK2V617F to regulate specific miRNAs, lineage negative (lin-) murine marrow progenitor cells were transduced with JAK2V617F or empty vector, allowed to form colonies for 7 days and miRNA levels in the colonies were determined. Again miR-200a, miR-9 and miR-22* and miR-155 were responsive to JAKV617F overexpression, while mir-134 was not. Transduction of lineage negative murine marrow progenitor cells with a lentiviral vector harboring mir-155 yielded a 30% increase in a myeloid colony formation in vitro. The effect is consistent with the reported ability of mir-155 to induce myeloproliferation in mice. Transduction of marrow progenitors with miR-133b, which is activated in MPN patients, responsive to JAK2V617F manipulation and not previously reported to have a role in hematopoiesis, led to an increase in both erythroid and myeloid colony formation. Taken together we conclude that at least 4 miRNAs are deregulated in CD34+ cells of MPN patients as a result of aberrant JAK2 activity. Two of these tested so far have a role in hematopoiesis. Part of the action of JAK2V617F in myeloproliferation may be mediated by specific miRNA, thus representing alternative therapeutic targets in MPN. Disclosures: Carroll: Sanofi Aventis Corp: Research Funding; Cephalon Oncoloy: Consultancy.

scholarly journals A pilot study of 3D tissue-engineered bone marrow culture as a tool to predict patient response to therapy in multiple myeloma

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kinan Alhallak ◽  
Amanda Jeske ◽  
Pilar de la Puente ◽  
Jennifer Sun ◽  
Mark Fiala ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Clinical Response ◽  
Ex Vivo ◽  
Drug Efficacy ◽  
Improve Patient Care ◽  
Hematologic Malignancies ◽  
Time Frame ◽  
Response To Therapy

AbstractCancer patients undergo detrimental toxicities and ineffective treatments especially in the relapsed setting, due to failed treatment attempts. The development of a tool that predicts the clinical response of individual patients to therapy is greatly desired. We have developed a novel patient-derived 3D tissue engineered bone marrow (3DTEBM) technology that closely recapitulate the pathophysiological conditions in the bone marrow and allows ex vivo proliferation of tumor cells of hematologic malignancies. In this study, we used the 3DTEBM to predict the clinical response of individual multiple myeloma (MM) patients to different therapeutic regimens. We found that while no correlation was observed between in vitro efficacy in classic 2D culture systems of drugs used for MM with their clinical efficacious concentration, the efficacious concentration in the 3DTEBM were directly correlated. Furthermore, the 3DTEBM model retrospectively predicted the clinical response to different treatment regimens in 89% of the MM patient cohort. These results demonstrated that the 3DTEBM is a feasible platform which can predict MM clinical responses with high accuracy and within a clinically actionable time frame. Utilization of this technology to predict drug efficacy and the likelihood of treatment failure could significantly improve patient care and treatment in many ways, particularly in the relapsed and refractory setting. Future studies are needed to validate the 3DTEBM model as a tool for predicting clinical efficacy.

scholarly journals O04. IN VITRO INCREASE IN REGULATORY T CELL NUMBERS BY ADALIMUMAB PREDICTS CLINICAL RESPONSE TO THERAPY IN PATIENTS WITH RHEUMATOID ARTHRITIS

Rheumatology ◽  
2017 ◽  
Vol 56 (suppl_2) ◽  
Author(s):  
Dao Xuan Nguyen ◽  
Laura Attipoe ◽  
Alice Cotton ◽  
Caroline Dore ◽  
Michael Ehrenstein
Keyword(s):  
Rheumatoid Arthritis ◽  
T Cell ◽  
Clinical Response ◽  
Regulatory T Cell ◽  
Response To Therapy ◽  
Cell Numbers

Knockdown of HCK Reduces Cell Death and Erythroid Differentiation in Human CD34+ Hematopoietic Progenitor Cells

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2860-2860
Author(s):  
Fernanda Marconi Roversi ◽  
Fernando Vieira Pericole ◽  
Adriana da Silva Santos Duarte ◽  
Karla Priscila Ferro ◽  
Flávia Adolfo Corrocher ◽  
...  
Keyword(s):  
Myeloid Leukemia ◽  
Specific Inhibitor ◽  
Erythroid Differentiation ◽  
Fold Change ◽  
Leukemic Cells ◽  
Leukemia Cells ◽  
Human Cd34 ◽  
Cd34 Cells

Abstract Myelodysplastic syndromes (MDS) are clonal disorders characterized by ineffective hematopoiesis and increased risk of transformation to acute myeloid leukemia (AML). The identification of genes and cellular pathways active in leukemia cells but not in normal hematopoietic stem/progenitor cells (HSC) may help to understand the key steps in the MDS and AML pathogenesis and lead to new approaches to further enhance the treatment of both diseases, considered incurable with non-transplantation therapy. Src kinase family (SFK) is a central mediator in multiple oncogenic signaling pathways and some SFK members (Hck, Lyn, Fgr, Fyn) had previously been described as overexpressed or activated in leukemic cells. However, to this moment, the role of hematopoietic cell kinase (HCK), the unique SFK member restricted expressed in hematopoietic cells, had not been characterized in MDS and AML pathogenesis as well as in HSC. In order to better understand the HCK importance in hematopoiesis, we used lentiviral shRNA vectors to knockdown the HCK expression in primary human CD34+ HSC. The HCK levels were reduced in approximately 70-80% (shHCK) compared to the control lentiviral shRNA (shControl-GFP). To promote erythroid differentiation, human CD34+ transduced cells were grown in methylcellulose for 7 days and in liquid media for another 6 days. During this experiment, shHCK cells showed decreased cell viability (fold change compared to shControl-GFP = 0.55, P<.0001, n=3) combined with an increase in CD71+ expression (fold change compared to shControl-GFP = 3, P<.01, n=3), indicating a delay in erythroid differentiation. As expected, shControl-GFP cells showed a decreased GATA1 expression during erythroid differentiation. Meanwhile, shHCK cells did not modulate GATA1 expression. Interestingly, without any stimulus, HCK knockdown in CD34+ cells significantly decreased apoptosis (AnnexinV+ cells) compared to shControl-GFP (fold change = 0.52, P<.01, n=4). Attempts have been made to overexpress HCK in CD34+ HSC, however more than 80% cells were apoptotic and further assays were not possible. Thus, in HSC, HCK participates of erythroid differentiation and apoptosis signaling. According to the HCK importance on HSC and that SFK inhibitors are undergoing early phase clinical testing, a specific inhibitory activity compound for HCK, named iHCK-37, had been developed by Dr Maurizzio Botta. We tested this compound on primary normal human CD34+ cells originated from healthy donors bone marrow samples and also from cord blood units. The iHCK-37 treatment did not change proliferation, survival and death of these normal CD34+ cells. Conversely, MDS and AML CD34+ cells treated with the same drug exhibited a dose-dependent growth inhibition. Likewise, following iHCK-37 treatment of MDS and AML total bone marrow mononuclear cells, the BFU-E and CFUs colony numbers were significantly decreased compared to untreated cells (vehicle). We also observed a potent in vitro antiproliferative activity of iHCK-37 against a panel of leukemia cell lines, with uM IC50 values in AML (5.0 - 5.8uM) and chronic myeloid leukemia (9.1 - 19.2uM). In addition, the combinatory in vitro treatment of iHCK-37 and 5-Azacitidine (Aza) also demonstrated additive effects relative to either drug alone. Interestingly, iHCK-37 or iHCK-37 plus Aza treatments of dysplastic and leukemia cells enhanced apoptosis and resulted in increased BAX and reduced BCL-XL protein levels. This result could be clinically relevant for MDS, as Aza is the only treatment available for higher-risk MDS, but with low response rates and frequent induced resistance and refractoriness over time. In summary, we herein have shown that HCK mRNA knockdown of normal CD34+ cells resulted in growth inhibition, decreased cell death and reduced erythroid differentiation, suggesting that HCK is essential for normal hematopoiesis. We presume that the deregulation of HCK pathway in leukemic cells might be crucial for MDS and AML pathogenesis. On the other hand, the inhibition of HCK protein activity with a specific inhibitor was able to restore the apoptotic pathways of leukemic cells, acting on cancer cells without alter any signaling of normal cells. Moreover, the specific inhibitor may have antineoplastic effect that can even be additive to current available drugs. Our study adds new insights to the role of HCK in MDS and AML as well as into potential new anticancer treatment strategies. Disclosures No relevant conflicts of interest to declare.

scholarly journals Cefiderocol for the Treatment of Adult and Pediatric Patients with Cystic Fibrosis and Achromobacter xylosoxidans Infections

2020 ◽  
Author(s):  
Nathaniel C Warner ◽  
Luther A Bartelt ◽  
Anne M Lachiewicz ◽  
Kathleen M Tompkins ◽  
Melissa B Miller ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Clinical Response ◽  
Pediatric Patients ◽  
Treatment Options ◽  
Achromobacter Xylosoxidans ◽  
Pre Treatment ◽  
Emergence Of Resistance

Abstract Treatment options for Achromobacter xylosoxidans are limited. Eight cystic fibrosis patients with A. xylosoxidans were treated with 12 cefiderocol courses. Pre-treatment in vitro resistance was seen in 3/8 cases. Clinical response occurred after 11/12 treatment courses. However, microbiologic relapse was observed after 11/12 treatment courses, notably without emergence of resistance.

scholarly journals Differential Expression of microRNAs and Transcription Factors in Primary Human Erythroblast Culture of β-Thalassemia Intermedia Patients

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2691-2691
Author(s):  
Regiane Ferreira ◽  
Carolina Lanaro ◽  
Kleber Yotsumoto Fertrin ◽  
Marcos André Cavalcanti Bezerra ◽  
Aderson da Silva Araujo ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Globin Gene ◽  
Erythroid Differentiation ◽  
Fold Change ◽  
High Expression ◽  
Pcr Array ◽  
Ineffective Erythropoiesis ◽  
Down Regulation ◽  
Cd34 Cells

Abstract Erythropoiesis is controlled by a complex interplay of signaling pathways, key transcription factors (TFs), as well as microRNAs (miRs). Although studies have identified miRs and TFs that regulate erythroid commitment and differentiation, the mechanism by which these molecules contribute to erythroid disorders, such as beta thalassemia (BT), is unclear. The aim of this study was to investigate miRs and TFs expression during in vitro erythroid differentiation in normal and ineffective erythropoiesis. Peripheral blood CD34+ cells were isolated from a transfusion-independent BT intermedia patient, homozygous for HBB IVS-I-6 (T→C), and cultured in a liquid system stimulating erythropoiesis. Cells were collected on day 10 for quantification of 84 mature miRs using a miRNA PCR Array platform. To minimize interindividual genetic variability, a control sample was collected from a healthy sibling (CON). For validation, CD34+ cells from 4 other BT patients and 4 healthy subjects were cultured and collected on days 7, 10, and 13 to measure miRs and TFs expressions during erythroid differentiation. Results: Data analysis from the miRNA PCR Array showed deregulated expression of 13 miRs in BT compared to CON: miRs 150-5p and 7-5p were decreased (fold change: -3.26 and -2.80) and miRs 32-5p, 96-5p, 155-5p, 146a-5p, 210, 125b-5p, 99a-5p, 125a-5p, 124-3p, 196b-5p, and 130a-3p were increased (fold change: 2.04; 3.72; 3.04; 2.27; 8.42; 2.82; 3.13; 2.48; 5.14; 2.00, and 3.07). The expression profile of ten miRs was successfully validated with the samples on day 10 of BT cultures. In addition, miRs 96-5p, 155-5p, and 210 remained increased on day 13 (P<0.05). Three miRs were down-regulated in BT cultures: 150-5p on days 10 and 13, miRs 24-3p and 144-3p only on day 13 (P<0.05). Seven TFs showed low expression in BT cultures: FOG1 and ALK4 on day 7 (P<0.05), NFE2 and LMO2 on day 10 (P<0.05), and GATA1, TAL1, and KLF1 on days 10 and 13 (P<0.05). Up-regulation of ETO2 occurred on days 10 and 13 (P<0.05), while cMYB, GATA2, LDB1, and PU.1 did not change significantly. Previous studies have shown that up-regulation of ETO2 was associated with low hemoglobin levels and its down-regulation contributes to the activation of TFs involved in GATA1-complex, such as LMO2, FOG1, and TAL1. Based on our preliminary results, the down-regulation of these TFs could be associated with the high expression of ETO2 in BT cultures. Additionally, the low levels of GATA1 may contribute with the decrease of miRs 24-3p and 144-3p, since it is known that this TF directly activates the transcription of these miRs. Our data are also supported by high expression of miRs 96-5p, 146a-5p, and 155-5p previously described in a cell culture model of induced ineffective erythroid maturation. In silico analysis predicted that miR-210 could target two regions of mKLF1, a critical transcription factor that directly activates β-globin and indirectly represses γ-globin gene expression. KLF1 down-regulation observed on days 10 and 13 of BT culture supports a hypothetical miR-210-mKLF1 interaction. Conclusion: Our data report associations between differentially expressed miRNAs and their possible targets during in vitro beta thalassemic erythroid differentiation. These results corroborate the importance of miRNAs in the hematopoietic process and highlight that further studies of the interaction between TFs and miRs may contribute to a better understanding of the pathophysiology of ineffective erythropoiesis. Support by FAPESP and CNPq Disclosures No relevant conflicts of interest to declare.

scholarly journals Inhibition of LSD1 in Myelodysplastic Syndrome Progenitors Restores Differentiation of CD141Hi Conventional Dendritic Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1695-1695
Author(s):  
Pragya Srivastava ◽  
Stephanie Tzetzo ◽  
Eduardo Cortes Gomez ◽  
Kevin Eng ◽  
Prashant K Singh ◽  
...  
Keyword(s):  
Clinical Trial ◽  
Dendritic Cells ◽  
Myelodysplastic Syndrome ◽  
Histone Modifications ◽  
Research Funding ◽  
Fold Change ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Pharmacologic Inhibition

Background: Immunotherapeutic approaches for myelodysplastic syndrome (MDS) show promise, but progress is limited by our incomplete understanding of the immunologic milieu. In a recent Phase I trial, we found that MDS patients with higher numbers of CD141Hi conventional dendritic cells (cDCs) were more likely to respond to NY-ESO-1 vaccination. In solid tumor models, the CD141Hi cDC is critical for initiating anti-tumor immune responses but its impact in myeloid malignancies is unknown. In studies of primary human specimens and mouse models, we tested the hypothesis that MDS patients exhibit decreased quantity and quality of CD141Hi cDCs due to impaired myeloid differentiation. Methods: Bone marrow (BM) cells were collected from MDS patients (pre-treatment) and age matched healthy donors (HD; defined as absence of hematologic malignancy). We quantified DC populations, stem, progenitor cells and interferon regulatory factor-8 (IRF-8) expression using flow cytometry and RT-qPCR. Histone modifications were assessed by chromatin immunoprecipitation. To assess DC differentiation of progenitors, human CD34+ and mouse c-kit+ cells were expanded and differentiated in vitro. Results: We found fewer CD141Hi cDCs (p<0.0001), CD1c+ cDCs (p<0.005) and plasmacytoid DCs (CD123+ pDCs; p<0.005) overall in BM samples from MDS patients (n=71) compared to HD (n=17). We stratified MDS patients based on the relative number of DCs and found that only those patients with highest number of CD141Hi cDCs had superior survival (p < 0.05). No differences in survival were seen in patients stratified by the CD1c+ cDCs (p = 0.96) and CD123+ pDCs (p = 0.32) populations. We hypothesized that decreased numbers of CD141Hi cDCs and adverse survival in MDS patients resulted from impaired differentiation of DC progenitors. We showed that MDS patients (n=19) have fewer monocyte-DC progenitors (MDP) and common DC progenitors (CDP) compared to HD (n=11; p<0.01). We then hypothesized that MDS progenitors express lower levels of IRF8, a master regulator of CD141Hi cDC differentiation. IRF8 expression was significantly lower in CDPs from MDS patients compared to HD (p<0.05). Furthermore, MDS patients with lower levels of IRF8 (n=8) in their MDPs showed a trend towards production of fewer CDPs and significantly fewer CD141Hi cDCs compared to those with higher levels of IRF8 (n=10; p<0.005). These results suggest that approaches to increase IRF8 expression could enhance CD141Hi cDC differentiation. We hypothesized that inhibition of lysine-specific histone demethylase 1A (LSD1), which increases IRF8 expression in myeloid leukemia cells, would induce CD141Hi cDC differentiation. Pharmacologic inhibition of LSD1 increased IRF8 expression (both mRNA and protein) in KG-1 cells, a model of human CD34+ cells, and in HD and MDS CD34+ progenitors (p<0.05). LSD1 inhibition in KG-1 cells resulted in increased H3K27 acetylation (3861-fold change) and H3K4 dimethylation (922.4-fold change) compared to PBS (p<0.05) at a region -70 kb to the IRF8 transcriptional start site, a putative regulatory element that demonstrated the highest level of LSD1 binding. These data indicate that LSD1 inhibition alters histone modifications at the IRF8 locus, resulting in increased expression of IRF8. Pharmacologic inhibition of LSD1 in HD CD34+ cells increased the number of mature CD141Hi cDCs in 92% of specimens (n = 12; 3.4 fold-change) compared to PBS. Similarly, LSD1 inhibition in MDS CD34+ cells increased the number of CD141Hi cDCs (n = 12) in 75% of patient specimens (16.8 fold-change) compared to PBS. IRF8 function is conserved between mice and humans. To test whether the effect of LSD1 inhibition on cDC differentiation was dependent on IRF8, we compared the effect of LSD1 inhibition on BM c-kit+ cells from Irf8 knock-out mice (Irf8-KO) and littermate controls (WT). LSD1 inhibition in WT c-kit+ cells resulted in increased numbers of CD141Hi cDCs in vitro (p<0.05). By contrast, LSD1 inhibition of Irf8-KO c-kit+ cells did not result in differentiation of CD141Hi cDCs. These data suggest that LSD1 inhibition drives CD141Hi cDCs differentiation through IRF8. Conclusion: These data reveal a previously unrecognized determinant of the immune microenvironment in MDS. The opportunity for epigenetic regulation of CD141Hi cDC differentiation in MDS offers an opportunity for intervention and a potential adjunct to immunotherapy for patients. Disclosures Sait: Celgene: Consultancy. Griffiths:Boston Scientific: Consultancy; Boston Scientific: Consultancy; Genentech, Inc.: Research Funding; Persimmune: Consultancy; Persimmune: Consultancy; Abbvie, Inc.: Consultancy; Genentech, Inc.: Research Funding; Novartis Inc.: Consultancy; Celgene, Inc: Consultancy, Research Funding; New Link Genetics: Consultancy; New Link Genetics: Consultancy; Astex Phramaceuticals/Otsuka Pharmaceuticals: Consultancy, Research Funding; Astex Phramaceuticals/Otsuka Pharmaceuticals: Consultancy, Research Funding; Novartis Inc.: Consultancy; Partner Therapeutics: Consultancy; Partner Therapeutics: Consultancy; Appelis Pharmaceuticals: Other: PI on a clinical trial; Onconova Therapeutics: Other: PI on a clinical trial; Appelis Pharmaceuticals: Other: PI on a clinical trial; Onconova Therapeutics: Other: PI on a clinical trial; Celgene, Inc: Consultancy, Research Funding; Abbvie, Inc.: Consultancy, PI on a clinical trial.

Characterization Of Bone Marrow Derived CD34+ Cells With Different Mobility Potentials By Micro RNA Fingerprinting

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4844-4844
Author(s):  
Blake Warbington ◽  
Daniel Weinstein ◽  
David Mallinson ◽  
Daria Olijnyk ◽  
Sarah Paterson ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Therapy ◽  
Mirna Expression ◽  
Phase Iii ◽  
Employment Equity ◽  
Cd34 Cells ◽  
Release Assay ◽  
Equity Ownership

Abstract Background AMR-001, an autologous CD34+ cell product derived from mini-marrow harvest, is currently undergoing Phase II trials to treat acute myocardial infarction (AMI). AMR-001 is administered to the patient by infusion via the infarct related artery within five to ten days following coronary artery stenting post AMI. At the time of infusion, it is believed that the infarct-region SDF-1 (stromal derived factor) levels are peaked and scar formation has not yet occurred. It was found that, in addition to the quantity of CD34+ cells infused, improvement in cardiac perfusion and infarct size correlated with the mobility potential of CD34+ cells mediated by a SDF-1 gradient (Quyyumi et al, Am Heart J 2011, 161:98–105). We have developed a cell based in vitro mobility assay as a potential potency release assay for AMR-001. However, this assay is not suitable for a Phase III or commercial scale release assay due to the length of the assay, high skill level required to perform, and variability. To develop a more robust assay, we have initiated a study to identify potential microRNAs (miRNAs) that may be used as biomarkers for CD34+ cell SDF-1 driven migration. Our preliminary results suggest CD34+ cells with different mobility potentials may be characterized by miRNA fingerprinting. Methods Cryopreserved purified CD34+ cells derived from bone marrow of healthy donors were purchased from a commercial vendor. Thawed CD34+ cells were washed and the cells were assayed in an in vitro transwell system (Jo et al, J Clin Invest 2000, 105:101-111). The trans-membrane migration of CD34+ cells into the lower chamber in the presence of SDF-1, as well as the non-mobilized CD34+ cells in the upper chamber, were collected after 4 hours incubation at 37°C. Total RNA of the cells was isolated and the miRNA expression profile was analyzed using SurePrint G3 Human v16 microRNA 8x60K microarray slide (Agilent, Santa Clara, CA). A normalization algorithm was used to generate miRNA expression profiles (SistemQC™, Sistemic, Ltd) for the characterization of untreated cells, the mobilized population that migrate towards SDF-1, and non-mobilized population; from two independent donors. Results Two hundred and four (204) miRNAs were reliably detected across the cell samples. The mobilized cells had different miRNA profiles compared with non-mobilized/untreated cells. Hierarchical cluster analysis showed that mobilized cells grouped separately from the non-mobilized/untreated cells. Conclusion Analysis of the miRNA profiles of the CD34+ cells across two independent donors, identified a number of key miRNAs (kmiRs™) that represent possible markers for a mobility phenotype. Additional samples will be analyzed to confirm these preliminary findings. This approach will enable the identification of markers associated with mobility potential of CD34+ cells and the potential development of a molecular biomarker assay for potency. Disclosures: Warbington: Progenitor Cell Therapy, LLC: Employment. Weinstein:Progenitor Cell Therapy, LLC: Employment. Mallinson:Sistemic, Ltd.: Employment, Equity Ownership. Olijnyk:Sistemic, Ltd.: Employment. Paterson:Sistemic, Ltd.: Employment. Ridha:Sistemic, Ltd.: Employment. O'Brien:Sistemic, Ltd.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees. Lin:Progenitor Cell Therapy, LLC: Employment. LeBlon:Progenitor Cell Therapy, LLC: Employment. Fong:NeoStem, Inc.: Employment. Chan:Progenitor Cell Therapy, LLC: Employment.

miRNA Predictors of Response to DNA Methyltransferase Inhibitors

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1913-1913
Author(s):  
Amma Bosompem ◽  
Yan Guo ◽  
Sanjay Mohan ◽  
Begum Erdogan ◽  
Muhammad Raza ◽  
...  
Keyword(s):  
Complete Remission ◽  
Small Rna ◽  
Mirna Expression ◽  
Dna Methyltransferase ◽  
Post Treatment ◽  
Response To Therapy ◽  
Hematopoietic Stem ◽  
Dna Methyltransferase Inhibitors ◽  
Pre Treatment ◽  
Normal Controls

Abstract Background Myelodysplastic syndrome (MDS) is a common disease of the elderly characterized by ineffective maturation of hematopoietic cells manifesting as low blood counts, morphologic atypia, and predisposition to the development of acute leukemia. The only FDA-approved drugs for the treatment of all subytpes of MDS are the DNA methyltransferase inhibitors (DNMTis), azacytidine and decitabine. However, only a minority of patients achieve a hematologic response or better on these agents. We have previously identified a miRNA signature which distinguishes MDS patients from normal controls. We sought to investigate whether miRNA expression might also be predictive of how patients respond to therapy with DNA methyltransferase inhibitors (DNMTis). Design We collected paired fresh frozen bone marrow mononuclear cells (BM-MNCs) from 28 patients with MDS both pre and post treatment with DNMTis as well as from 30 normal controls. Following enrichment for miRNAs and other small RNA species using the TruSeq Small RNA sample preparation kit (Illumina, San Diego, CA), miRNAs were sequenced on an Illumina HiSeq 2500. Alignment was performed using Bowtie 2 against reference sequences from mirBASE and differential expression analysis was analyzed using a rank sum score from the following analysis programs: DESeq, edgeR and baySeq. The response to the DNMTis was characterized based upon the following scale: 1 = complete remission, 2 = marrow complete remission, 3 = partial remission, 4 = hematologic improvement, 5 = stable disease, and 6 = progressive disease. Linear regression analysis was conducted using the response score as outcome and miRNA expression value as the predictor. Results Several differences were found between the control samples and the pre-treatment MDS samples, many of which have been described previously, including hsa-miR-125b, hsa-miR-342, hsa-miR-140, and hsa-miR-150. Not unexpectedly, the expression of numerous miRNAs was significantly altered as a result of treatment with DNMTis. However, there were no significant miRNA expression differences between responders and non-responders in the post-treatment samples and no significant differences between the changes in miRNA expression (pre versus post treatment changes) in responders and non-responders. Interestingly, 5 miRNAs demonstrated significant association between their pre-treatment expression and the degree of response (FDR < 0.05): hsa-miR-125a, hsa-miR-342, hsa-miR-146b, hsa-miR-146a, hsa-miR-423, and hsa-miR-150. These miRNAs share striking overlap in their mRNA targets including key pathway regulators in the p53 pathway as well as numerous growth factors implicated in hematopoietic stem cells as well as hematopoietic stem cell markers. Conclusion These studies confirm that miRNA expression differences differentiate between MDS and normal controls. The administration of DNMTis results in widespread expression changes regardless of the response to therapy. However, the expression of a small handful of miRNA are positively correlated with the degree of response in MDS pre-treatment samples to DNMTis and therefore may be useful as markers which predict responders to DNMTi therapy in addition to suggesting biological pathways implicated in the mechanism of response. These results await further prospective studies for confirmation. Disclosures No relevant conflicts of interest to declare.

Presence and Possible Origin of ɛ(γ-Glutamyl)Lysine Isodipeptide in Human Plasma

1992 ◽  
Vol 67 (01) ◽  
pp. 060-062 ◽  
Author(s):  
J Harsfalvi ◽  
E Tarcsa ◽  
M Udvardy ◽  
G Zajka ◽  
T Szarvas ◽  
...  
Keyword(s):  
Human Plasma ◽  
Fibrinolytic Therapy ◽  
Normal Human Plasma ◽  
Normal Human ◽  
Hplc Technique ◽  
Significant Change

Summaryɛ(γ-glutamyl)lysine isodipeptide has been detected in normal human plasma by a sensitive HPLC technique in a concentration of 1.9-3.6 μmol/1. Incubation of in vitro clotted plasma at 37° C for 12 h resulted in an increased amount of isodipeptide, and there was no further significant change when streptokinase was also present. Increased in vivo isodipeptide concentrations were also observed in hypercoagulable states and during fibrinolytic therapy.

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