scholarly journals Establishing a Novel in Vitro Informed Precision Clinical Trial Pathway for Refractory Pediatric Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2633-2633
Author(s):  
Chunfen Zhang ◽  
Ritul Sharma ◽  
Thakur Satbir ◽  
Allison Cheney ◽  
Olena Vaske ◽  
...  
Keyword(s):  
Gene Expression ◽  
Clinical Trial ◽  
Bone Marrow ◽  
Cell Line ◽  
Leukemic Cells ◽  
Parp Cleavage ◽  
Growth And Survival ◽  
Molecular Pathway ◽  
Cells In Culture

Introduction: In children diagnosed with leukemia, relapse and its associated morbidity and mortality remain the most dreaded consequences of the disease. Therefore, the discovery and implementation of novel and broadly applicable therapeutic strategies for these patients are urgently needed. Currently, a number of precision therapeutic approaches have been formulated where molecular analyses of the malignant cells have been used to inform, often multiple, probable targets and potential therapeutic agents. However, a common drawback of this approach has been the uncertainty involved in selecting the drug with the most and clinically relevant cytotoxic potential. In vitro xenograft approaches, although can provide key information on drug activity and side effects, are time consuming and impractical and cumbersome in most cases. We have recently demonstrated the ability of a bone marrow stromal derived cell line to sustain the growth and survival of patient leukemic cells in culture that has allowed in vitro evaluations of drug response.This methodology was combined with a previously validated molecular pathway analysis program to identify effective agents or combinations for a subsequent informed precision clinical trial. Methods: Gene expression profiles from refractory pediatric leukemic cells were analyzed against similar data from more than 12, 000 tumors and outlier analyses were carried out to generate a list of overexpressed genes. This information was computed to identify hypothetically activated pathways, druggable targets and potential agents from a panel of FDA approved drugs. A bone marrow stromal cell line was established and characterized that has been shown to support leukemia cell proliferation in vitro. Briefly, stromal cells were co-cultured with leukemic cells at pre-determined ratios with and without the drugs identified in the genomic analysis. After four days in culture, leukemic cells were re-suspended and analyzed for proliferation. Target modulation and activated cell death pathways were queried by Western blot analyses. Results: Multiple targets and potential agents for effective therapeutics were identified against an initial set of relapsed leukemia specimens. For example, in patient # P700491 (pre B-ALL) gene expression data sets revealed clustering within the area of ALL and AML in the reference cancer genomics data. Comparative tumor RNA seq outlier analysis showed molecular abnormalities in BTK, JAK3 and PIK3CD, corresponding to molecular categories of RTK, JAK-SAT and PI3K-AKT-mTOR pathways targetable by the drugs Ibrutinib, WHI-P131 and Idelalisib, respectively. However, in vitro studies showed significant cell killing with Idelalisib and not with the other two agents. Target modulation assays showed effective induction of apoptosis including PARP cleavage in Idelalisib treated leukemia cells compared to controls, indicating the feasibility of this approach to effectively identify potentially applicable agents for this individual patient. Conclusions: We demonstrate the ability of a newly cloned bone marrow stromal derived cell line to sustain the growth and survival of patient leukemic cells in culture that has allowed in vitro target modulation and target validation analyses for cytotoxicity. This methodology was combined with a previously demonstrated molecular pathway interrogation program to ascertain effective agents or combinations for an experimentally informed precision clinical trial. Importantly, our data showed that genomically identified actionable targets are not universally predictive of tumor response and an in vitro cytotoxicity analysis step may enhance the accuracy of this approach. We describe the practical advantages and versatility of this work-flow to inform the selection of agents in future umbrella trials. It is anticipated that the information obtained will lead to an applicable clinical trial the near future. Disclosures Narendran: Bayer: Honoraria, Other: CANTRK Advisory Board .

The Multi-Targeted Receptor Tyrosine Kinase Inhibitor, ABT-869, Induces Apoptosis of AML Cells Both In Vitro and In Vivo.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 616-616 ◽  
Author(s):  
Deepa B. Shankar ◽  
Jenny C. Chang ◽  
Bertrand Parcells ◽  
Salemiz Sandoval ◽  
Junling Li ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Tyrosine Kinase ◽  
Cell Line ◽  
Progenitor Cells ◽  
Cell Lines ◽  
Receptor Tyrosine Kinase ◽  
Scid Mice ◽  
Parp Cleavage

Abstract Children with acute myeloid leukemia (AML) have less than 60% overall survival despite aggressive chemotherapy and bone marrow transplantation. Only one third of the adult patients diagnosed with AML will be cured. AML blast cells from up to 30% of patients express a constitutively active receptor tyrosine kinase, FLT3-ITD, which contains an internal tandem duplication in the juxtamembrane domain. Patients with FLT3-ITD have a worse prognosis. ABT-869 is a novel multi-targeted small molecule inhibitor of receptor tyrosine kinases and is a potent inhibitor of FLT3, c-Kit, and all members of the VEGF and PDGF receptor families. To determine the effects of ABT-896 on AML cells, we treated AML cell lines, primary cells, and tumors in xenograft models with varying concentrations of the drug. In vitro viability assays showed that ABT-869 inhibited the growth of two different cell lines, MV-4-11 (human AML cell line that expresses FLT3-ITD) and BAF3-ITD (murine B-cell line stably transfected with the FLT3-ITD) at an IC50 of 10nM. ABT-869 was also effective against another mutation of FLT3, D835V, but at higher concentrations (IC50 of 100nM). Phosphorylation of FLT3 and activation of downstream signaling molecules, STAT5 and ERK, were inhibited by ABT-869 in a concentration-dependent manner. Cells were also stained with Annexin V-FITC and Propidium Iodide, and analyzed using FACS. ABT-869 induced apoptosis, caspase-3 activation, and PARP cleavage after 48 hours. To examine the in vitro effects of ABT-869 on normal hematopoietic progenitor cells, we performed methylcellulose-based colony assays with human bone marrow. No significant difference was observed in the number and type of colonies formed using BM cells treated with ABT-869 or control, up to a concentration of 1 micromolar. These results suggest that ABT-869 is not toxic to normal bone marrow progenitor cells at concentrations that are effective against AML cells. To examine the effects of ABT-869 in vivo, we treated SCID mice injected with MV-4-11, Baf3-ITD, Baf3-D835V, or Baf3-WT cells, with oral preparations of ABT-869. Complete regression of MV-4-11 tumors was observed in mice treated with ABT-869 at 20 and 40 mg/kg/day. No adverse effects were detected in the peripheral blood counts, bone marrow, spleen or liver. Histology of the tumors from the control-treated group showed a high degree of proliferation by Ki-67 staining, increased mitotic figures, and a well-defined tumor mass. In contrast, the tumors from mice treated with ABT-869 showed a number of apoptotic bodies by TUNEL staining and the presence of reactive, inflammatory cells. Interestingly, we also observed that mice that received ABT-869 the day after injection of AML cells remained tumor-free for over 2 months in contrast to the mice receiving the vehicle alone. Inhibition of FLT3 phosphorylation was demonstrated in the tumors from mice treated with ABT-869. We are evaluating the activity of ABT-869 treatment of SCID mice injected with Baf3-ITD, Baf3-D835V, or Baf3-WT cells. NOD-SCID mouse models are currently being used to analyze the effects of ABT-869 on primary AML cells in vivo. Our preclinical studies demonstrate that ABT-869 is effective and nontoxic, and provide rationale for the treatment and prevention of relapse in AML patients.

scholarly journals Phenotypic heterogeneity among stromal cell lines from mouse bone marrow disclosed in their extracellular matrix composition and interactions with normal and leukemic cells

Blood ◽  
1985 ◽  
Vol 66 (2) ◽  
pp. 447-455 ◽  
Author(s):  
D Zipori ◽  
J Toledo ◽  
K von der Mark
Keyword(s):  
Bone Marrow ◽  
Extracellular Matrix ◽  
Cell Line ◽  
Cell Lines ◽  
Stromal Cell ◽  
Leukemic Cells ◽  
Matrix Composition ◽  
Mouse Bone Marrow ◽  
Type I

Abstract Study of a series of stromal cell lines from mouse bone marrow (MBA) verified and extended their classification as phenotypically distinct subtypes. Production of extracellular matrix proteins was examined using specific antibodies. Fibronectin and laminin were detected in all of the cell lines tested, yet 14F1.1 adipocytes exhibited particularly prominent extracellular deposition. This cell line and MBA-13.2 cells were positive to both collagen types I and IV, whereas MBA-1 and MBA- 2.1 were stained with anticollagen type I antibodies only. Coculture experiments revealed differences among the lines in their effects on normal myeloid cells and leukemic cell lines. In promoting the in vitro accumulation of myeloid progenitors (CFU-C), 14F1.1 cells surpassed the others. The MBA-2.1 cell line was particularly inhibitory to MPC-11 plasmacytoma and Friend erythroleukemia cells. However, the latter were refractory to other stromal cell lines, whereas MPC-11 cells were inhibited to various degrees by virtually all of the cell lines. Physical separation between the interacting cells reduced the inhibition in some but not all cases, and no inhibitory activity was detected in conditioned media. The MBA-13 stromal cells synergistically promoted the differentiation of dimethylsulfoxide (Me2SO)-induced Friend erythroleukemia. The latter cells themselves, at high concentrations, as well as some of the stromal cell lines and unrelated adherent cells, antagonized the Me2SO effect, revealing possible reversible stages in the Friend cell differentiation pathway.

TEL-AML1 Affects the Regulation of Cytoskeleton and Causes Alteration In Cellular Adhesive and Migratory Properties In An In Vitro Model of Pre-Leukemia.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3624-3624
Author(s):  
Chiara Palmi ◽  
Grazia Fazio ◽  
Ilaria Brunati ◽  
Valeria Cazzaniga ◽  
Valentina André ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Line ◽  
Conflicts Of Interest ◽  
Fusion Gene ◽  
Expression System ◽  
Leukemic Cells ◽  
Calcium Flux ◽  
Actin Dynamics ◽  
Childhood Leukaemia

Abstract Abstract 3624 Introduction: The t(12;21) chromosome translocation generating TEL-AML1 chimeric fusion gene is a frequent initiating event in childhood leukaemia. Its impact is to generate a clone of covert, clinically silent pre-leukemic B cell progenitors. The leukemia arises only following second, post-natal hit/genetic events occurring years later. Moreover, relapse of leukemia is frequently arising from the pre-leukemic clone. Aim of our study is to investigate how TEL-AML1 expression can sustain this covert condition for many years. In a recent paper we described that the fusion gene rendered the B precursors resistant to the inhibitory activity of TGFbeta. Here we want to inquire into other factors that can explain the positive selection of the pre-leukemic clones over the normal counterpart. In particular, given the importance of the interaction with the microenvironment for survival signals for normal and leukemic stem cells, we question if the fusion gene causes changes in cellular adhesive and migratory properties. Methods: the study was performed by using two different models: i) a TEL-AML1 inducible expression system on the murine pro-B Ba/F3 cell line and ii) murine primary B lymphocytes (pre-BI cells) isolated from fetal liver, stably transduced with the pMIGR1-TEL-AML1-IRES-GFP construct. Gene expression assays were performed by using TaqMan (Applied Biosystems) and PCR Array technologies (SABioscences). Results: The expression of TEL-AML1 in Ba/F3 cell line causes over-expression of genes regulators of the cytoskeleton, specifically involved in cellular movement and in the regulation of actin dynamics. This gene expression alteration results in changes in the cellular morphology and phenotype: the cells acquire long extensions and several molecules involved in cell adhesion and migration are disregulated. Moreover, the TEL-AML1 positive cells present an increased ability to adhere to the ICAM1 substrate, but they also show a significant defect in the chemotactic response to CXCL12 in transwell migration assays in vitro, although the expression and the recycling of CXCR4 receptor are unaffected. This inability is not due to defects to migrate in general, as spontaneous motility is enhanced, but it is associated with a defect in CXCR4 signaling. In particular, CXCL12 calcium flux and ERK phosphorylation were inhibited. Those results have been confirmed in murine PreBI primary cells. Conclusions: in our murine models, TEL-AML1 affects the cytoscheleton regulation and causes alteration in cellular adhesive and migratory properties. We are now investigating how these alterations can give advantages to the pre-leukemic cells in the pathogenesis of TEL-AML1–expressing leukemia. Disclosures: No relevant conflicts of interest to declare.

scholarly journals PHF6 Restricts AML Acceleration By Promoting Myeloid Differentiation Genes in Leukemic Cells

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 42-43
Author(s):  
Sapana S Jalnapurkar ◽  
Aishwarya Pawar ◽  
Patrick Somers ◽  
Gabrielle Ochoco ◽  
Subin S George ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Line ◽  
Median Survival ◽  
Point Mutations ◽  
Leukemic Cells ◽  
Myeloid Differentiation ◽  
Molecular Function ◽  
Self Renewal

Acute myeloid leukemia is caused by the accumulation of mutations in hematopoietic stem and myeloid progenitor cells, resulting in increased self-renewal, inhibition of differentiation, and aberrant proliferation. Although genomic studies have comprehensively identified genes that are mutated in acute leukemias, the functional roles of many of them, and the consequences of their mutations, remain poorly understood. PHF6 (PHD-finger protein 6) is an X-linked gene that is mutated in 3.2% of de novo AML, 4.7% CMML, 3% MDS, and 1.6% CML patients. Two-thirds of somatic mutations in PHF6 are frameshift and nonsense mutations distributed throughout the gene body, resulting in loss of PHF6 protein. One-third of the mutations are point mutations clustered in the ePHD2 (extended PHD) domain, and the consequence of these mutations on PHF6 function is unknown. The functional role of PHF6 and the mechanism by which PHF6 mutations accelerate AML has not yet been determined. In this study, we delineate the cellular and molecular function of PHF6 in AML using in vitro and in vivo models. In agreement with recently published reports, we found that pan-hematopoietic deletion of Phf6 using the Vav-Cre recombinase system gave competitive transplantation advantage to HSCs, with sustained multi-lineage reconstitution without exhaustion over three rounds of serial transplantations, demonstrating that Phf6 represses HSC self-renewal. However, loss of Phf6 alone was insufficient to cause hematopoietic malignancy in the mouse model when monitored for one year. To determine the function of PHF6 in AML progression, we transduced cKO (Vav-Cre; Phf6 flox) or WT (Vav-Cre only) bone marrow cells with MSCV retrovirus expressing HOXA9 (WT+HOXA9 and cKO+HOXA9), and transplanted into irradiated recipient mice. The resulting HOXA9-driven AML was greatly accelerated in the Phf6 cKO background, with recipient mice succumbing faster (median survival 119 days) as compared to recipients transduced with HOXA9-transduced WT cells (median survival >180 days, p=0.003) (Fig 1A). This was also reflected by an increase in the number of circulating immature leukemic cells in peripheral blood at earlier timepoints. HOXA9-transduced cKO cells showed higher serial replating ability in an in vitro colony forming assay as compared with HOXA9-transduced WT cells (Fig 1B). We further investigated the molecular function of PHF6 using the THP-1 human AML cell line. PHF6 is a chromatin-binding protein with two ePHD domains, and its binding partners and pattern of chromatin occupancy are unclear. Using ChIP-Seq, we identified that PHF6 occupies enhancers, and its peaks show striking alignment with the peaks of the key myeloid transcription factors (TFs) RUNX1, PU.1, and IRF8 (Fig 1C). To assess the effect of the clinically relevant point mutation R274Q (in the ePHD2 domain) on the transcriptional effects produced by PHF6, we first generated a PHF6 KO clone from the THP-1 cell line, and then re-expressed either WT PHF6 or R274Q-mutant PHF6 in this KO line. Re-expression of WT PHF6 rescued the extensive gene expression changes produced by its knockout, but R274Q-mutant PHF6 was unable to produce any gene expression changes, indicating that it is a "transcriptionally dead" mutant (Fig 1D). Gene Ontology analysis of transcriptome changes induced by WT PHF6 showed that PHF6 promotes the expression of myeloid differentiation gene sets. In summary, PHF6 restricts AML progression by binding enhancers with key myeloid TFs, and promoting the expression of myeloid differentiation genes. R274Q mutation renders PHF6 unable to exert any downstream expression changes, indicating that the ePHD2 domain (where R274 is located, clustered with other amino acids showing point mutations in hematopoietic malignancies) is critical for PHF6 function, and likely mediates important functional interactions with chromatin partners. Our future work will involve dissection of the sequence of molecular events governed by PHF6 following enhancer occupancy, and the role of PHF6 in repressing AML self-renewal and promoting differentiation. Disclosures No relevant conflicts of interest to declare.

In Vitro Characterization of R14W Mutation in SEC23B, the CDAII Causative Gene

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2098-2098
Author(s):  
Maria Rosaria Esposito ◽  
Roberta Russo ◽  
Annaelena Troiano ◽  
Immacolata Andolfo ◽  
Roberta Asci ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Cell Line ◽  
Cellular Localization ◽  
Band 3 ◽  
Red Cell ◽  
Causative Gene ◽  
In Vitro Characterization

Abstract Abstract 2098 Congenital dyserythropoietic anemias (CDAs) designate a group of genetic disorders in which ineffective erythropoiesis is the predominant mechanism of anemia marked by distinct morphologic abnormalities of the majority of erythroblasts in the bone marrow. CDA type II (CDAII) is the most common type of CDA. It is characterized by a recessive model of inheritance, mild to moderate anemia, jaundice, and splenomegaly (Fukuda MN, Glycobiology 1990; Fukuda MN, Clin Haematol 1993), by the presence of bi- and multinucleated erythroblasts in bone marrow, with nuclei of equal size and DNA content, suggesting a cytokinesis disturbance (Schwarz K and Iolascon A. et al., Nat Genet. 2009). The specific hallmark of diagnosis is the presence of the more abundant protein of membrane red cell, band 3, in a hypoglycosilated state; this is thinner and migrated slightly faster than in controls on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Anselstetter V et al., Br J Haematol 1977). The causative gene of CDAII, SEC23B, is a member of the SEC23 subfamily of the SEC23/SEC24 family, which is involved in vesicle trafficking. The encoded protein has similarity to yeast Sec23p (66.4%) component of COPII, the coat protein complex responsible for vesicle budding from the ER. The function of this gene product has been implicated in cargo selection and concentration. The SEC23B gene spans approximately 54 kb on human chromosome 20p11.23 and codifies for a protein of 767 aminoacids divided in five functional domains: zink finger, trunk, β-sheet, helical and gelsolin domain. Although most of the SEC23B mutations are sporadic events, 4 mutations (R14W, E109K, R497C, I318T) accounted for more than 50% of mutated alleles. The aim of this study is the in vitro characterization of the R14W mutation, the most frequent variant in Italy, particularly in South of Italy (Russo R et al., Am J Hematol. in press). We used the human erythroleukemia cell line, HEL, as in vitro model because it is more similar to mature red cell. By using in silico tool ESyPred3D Web Server 1.0, we predicted that this aminoacidic substitution alters the zink finger domain 3D structure, when compared to wild type protein. This tool implements homology modeling approach followed by a final analysis with MODELLER release 4 in order to build a 3D model of the submitted protein (Lambert et al, 2002). However, when we transfected the SEC23B-R14W we observed a strong reduction of gene expression in the mutant when compared to SEC23B-wt construct by qRT-PCR. These results have been also confirmed at the protein level. In fact the protein expression of SEC23B-R14W showed a reduction comparable to gene expression respect to SEC23B-wt construct. Immunofluorescence analyses by confocal microscopy, were used for the investigation of the cellular localization of SEC23B-R1W protein and, interestingly, the localization of mutant protein was not changed when compared to that wt. Our data allow us to hypothesize that the mutation R14W gives rise to anomalous protein product quantity, but the protein function would be like not altered. Our findings demonstrated that the most frequent mutation found in Italy, SEC23B-R14W, results in a reduced half-life of the mutated mRNA, without altering the cellular localization in HEL cell line. SEC23B belongs to a multiproteic compelx that assembles with the others complex proteins in accordance with a specific structure. Each structure establishes a cargo selectivity. Further studies are necessary in order to understand what is the role of SEC23B in selectivity of the cargo in erythroid cells and how its disruption could determines the appearance of the principal pathological phenotype in CDAII patients, for example the hypoglycosilation of band 3. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Phenotypic heterogeneity among stromal cell lines from mouse bone marrow disclosed in their extracellular matrix composition and interactions with normal and leukemic cells

Blood ◽  
1985 ◽  
Vol 66 (2) ◽  
pp. 447-455
Author(s):  
D Zipori ◽  
J Toledo ◽  
K von der Mark
Keyword(s):  
Bone Marrow ◽  
Extracellular Matrix ◽  
Cell Line ◽  
Cell Lines ◽  
Stromal Cell ◽  
Leukemic Cells ◽  
Matrix Composition ◽  
Mouse Bone Marrow ◽  
Type I

Study of a series of stromal cell lines from mouse bone marrow (MBA) verified and extended their classification as phenotypically distinct subtypes. Production of extracellular matrix proteins was examined using specific antibodies. Fibronectin and laminin were detected in all of the cell lines tested, yet 14F1.1 adipocytes exhibited particularly prominent extracellular deposition. This cell line and MBA-13.2 cells were positive to both collagen types I and IV, whereas MBA-1 and MBA- 2.1 were stained with anticollagen type I antibodies only. Coculture experiments revealed differences among the lines in their effects on normal myeloid cells and leukemic cell lines. In promoting the in vitro accumulation of myeloid progenitors (CFU-C), 14F1.1 cells surpassed the others. The MBA-2.1 cell line was particularly inhibitory to MPC-11 plasmacytoma and Friend erythroleukemia cells. However, the latter were refractory to other stromal cell lines, whereas MPC-11 cells were inhibited to various degrees by virtually all of the cell lines. Physical separation between the interacting cells reduced the inhibition in some but not all cases, and no inhibitory activity was detected in conditioned media. The MBA-13 stromal cells synergistically promoted the differentiation of dimethylsulfoxide (Me2SO)-induced Friend erythroleukemia. The latter cells themselves, at high concentrations, as well as some of the stromal cell lines and unrelated adherent cells, antagonized the Me2SO effect, revealing possible reversible stages in the Friend cell differentiation pathway.

scholarly journals Effect of Nicotinamide, 1-Methylnicotinamide, and N'-Methylnicotinamide on Erythroid Colony Formation and γ-Globin Expression in Cultured Baboon CD34+ Cells

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 4-5
Author(s):  
Donald Lavelle ◽  
Vinzon Ibanez ◽  
Kestis Vaitkus ◽  
Yogenthiran Saunthararajah ◽  
Robert E. Molokie
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Cell Line ◽  
Globin Gene ◽  
Erythroid Differentiation ◽  
Cd34 Cells ◽  
Murine Erythroleukemia ◽  
Globin Gene Expression ◽  
In Cells

Pharmacological treatments designed to increase Fetal Hemoglobin (HbF) levels offer great promise to alleviate the symptoms and improve the lifespan of the vast numbers of patients afflicted with sickle cell disease (SCD) and β-thalassemia. Hydroxyurea can increase HbF, but a large fraction of patients with SCD do not respond to the drug. DNMT1 and LSD1 inhibitors are the most powerful drugs to increase HbF but are limited by side effects that include neutropenia, thrombophilia and/or thrombocytopenia. The development of new, more effective, and safer pharmacological strategies to augment HbF levels in the blood thus continues to be an important goal. Previous studies have shown that γ-globin gene expression is dynamically regulated during erythroid differentiation (Papayannopoulou et al PNAS 74:2923-2927, 1977). The proportion of γ-globin gene expression is higher at earlier stages (BFUe) of erythroid differentiation than at more advanced stages (CFUe). Therefore, we suggest the hypothesis that expansion of primitive, less differentiated progenitors might favor increased γ-globin, particularly when combined other HbF-inducing drugs. To investigate this hypothesis, we have tested whether nicotinamide (NAM), the major NAM metabolite 1-methylnicotinamide (1-mNAM), and N'-methylnicotinamide (N'-mNAM), a chemical derivative of NAM, can foster expansion of erythroid colony-forming cells (BFUe and CFUe) and increase γ-globin expression of cultured baboon CD34+ cells. Previous observations have shown that NAM facilitates in vitro expansion of cord blood CD34+ cells and enhanced long term engraftment in transplanted recipients (Horwitz et al J Clin Invest 124:3121, 2014). Contrasting effects of NAM, 1-mNAM, and N'-mNAM on differentiation and proliferation of the murine erythroleukemia cell line (MEL) have been previously reported (Terada et al PNAS 76:6414, 1979; Kuykendall et al Toxicol In Vitro 21:1656, 2007). While both NAM and N'-mNAM induced MEL cell differentiation, N'-mNAM was far more potent. In contrast, 1-mNAM increased cell proliferation, reduced spontaneous differentiation, and blocked differentiation induced by NAM and N'-mNAM. The effect of all three forms of NAM was examined using bone marrow (BM) CD34+ cells from a pre-clinical non-human primate large animal model. To test the effect of NAM, 1-mNAM, and N'-mNAM on expansion of erythroid colony-forming cells, the agents (5mM) were added to liquid cultures of baboon CD34+ bone marrow cells previously expanded for 5 days in serum-free expansion media (SFEM). Colony assays were performed on d8. In two experiments total erythroid colonies (BFUe and CFUe) were 2 fold higher in cultures treated with 1-mNAM compared to untreated controls (p<0.05) while no effect was observed in cultures treated with NAM. No colonies were observed in cultures treated with N'-mNAM (Figure 1A). Observation of Wright's stained cytospin preparations showed extensive erythroid differentiation on d8 in cells treated with N'-mNAM (Figure 1B). The effect of NAM, 1-mNAM, and N'-NAM on γ-globin expression was tested in baboon CD34+ cells grown in co-culture with the AFT024 cell line. NAM, 1-mNAM, or N'-mNAM (5mM) were added to cultures on d7. Expression of γ- and β-globin mRNA was measured by RT-PCR on d17. Increased γ-globin expression (0.57±0.04 γ/γ+β)) was observed in cells treated with N'-mNAM on d7 compared to untreated controls (0.20±0.09 γ/γ+β; p<0.001). NAM and 1-mNAM had no significant effect on γ-globin gene expression (Figure 1C). These results thus show that while erythroid colonies are increased by 1-mNAM, N'-mNAM is a potent inducer of erythroid differentiation and increases γ-globin expression in primary cultures of baboon CD34+ cells. In conclusion, 1-mNAM and N'-mNAM have contrasting effects on erythroid differentiation in primary baboon CD34+ cell cultures, confirming previous experiments in the MEL cell line. Future experiments are planned to test the effect of these agents on HbF in the baboon. Disclosures Saunthararajah: EpiDestiny: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

scholarly journals Colony formation in vitro by leukemic cells in acute lymphoblastic leukemia (ALL)

Blood ◽  
1978 ◽  
Vol 52 (4) ◽  
pp. 712-718 ◽  
Author(s):  
SD Smith ◽  
EM Uyeki ◽  
JT Lowman
Keyword(s):  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
Bone Marrow Cells ◽  
Lymphoblastic Leukemia ◽  
Lymphoid Cells ◽  
Assay System ◽  
Leukemic Cells ◽  
Specific Cell ◽  
Specific Cell Surface

Abstract An assay system in vitro for the growth of malignant lymphoblastic colony-forming cells (CFC) was established. Growth of malignant myeloblastic CFC has been previously reported, but this is the first report of growth of malignant lymphoblastic CFC. Established assay systems in vitro have been very helpful in elucidating the control of growth and differentiation of both normal and malignant bone marrow cells. Lymphoblastic CFC were grown from the bone marrow aspirates of 20 children with acute lymphoblastic leukemia. Growth of these colonies was established on an agar assay system and maintained in the relative hypoxia (7% oxygen) of a Stulberg chamber. The criteria for malignancy of these colonies was based upon cellular cytochemical staining characteristics, the presence of specific cell surface markers, and the ability of these lymphoid cells to grow without the addition of a lymphoid mitogen. With this technique, specific nutritional requirements and drug sensitivities can be established in vitro, and these data may permit tailoring of individual antileukemic therapy.

Overexpression of hTLX3 in Association with Mutant IL7Rα Is Sufficient to Generate T-ALL In Vivo and to Transform Thymocytes in Vitro

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 21-21
Author(s):  
Gisele Olinto Libanio Rodrigues ◽  
Julie Hixon ◽  
Hila Winer ◽  
Erica Matich ◽  
Caroline Andrews ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Cell Line ◽  
Lymphoblastic Leukemia ◽  
T Cell Leukemia ◽  
Cell Leukemia ◽  
Cell Counts

Mutations of the IL-7Rα chain occur in approximately 10% of pediatric T-cell acute lymphoblastic leukemia cases. While we have shown that mutant IL7Ra is sufficient to transform an immortalized thymocyte cell line, mutation of IL7Ra alone was insufficient to cause transformation of primary T cells, suggesting that additional genetic lesions may be present contributing to initiate leukemia. Studies addressing the combinations of mutant IL7Ra plus TLX3 overexpression indicates in vitro growth advantage, suggesting this gene as potential collaborative candidate. Furthermore, patients with mutated IL7R were more likely to have TLX3 or HOXA subgroup leukemia. We sought to determine whether combination of mutant hIL7Ra plus TLX3 overexpression is sufficient to generate T-cell leukemia in vivo. Double negative thymocytes were isolated from C57BL/6J mice and transduced with retroviral vectors containing mutant hIL7R plus hTLX3, or the genes alone. The combination mutant hIL7R wild type and hTLX3 was also tested. Transduced thymocytes were cultured on the OP9-DL4 bone marrow stromal cell line for 5-13 days and accessed for expression of transduced constructs and then injected into sublethally irradiated Rag-/- mice. Mice were euthanized at onset of clinical signs, and cells were immunophenotyped by flow cytometry. Thymocytes transduced with muthIL-7R-hTLX3 transformed to cytokine-independent growth and expanded over 30 days in the absence of all cytokines. Mice injected with muthIL7R-hTLX3 cells, but not the controls (wthIL7R-hTLX3or mutIL7R alone) developed leukemia approximately 3 weeks post injection, characterized by GFP expressing T-cells in blood, spleen, liver, lymph nodes and bone marrow. Furthermore, leukemic mice had increased white blood cell counts and presented with splenomegaly. Phenotypic analysis revealed a higher CD4-CD8- T cell population in the blood, bone marrow, liver and spleen compared in the mutant hIL7R + hTLX3 mice compared with mice injected with mutant IL7R alone indicating that the resulting leukemia from the combination mutant hIL7R plus hTLX3 shows early arrest in T-cell development. Taken together, these data show that oncogenic IL7R activation is sufficient for cooperation with hTLX3 in ex vivo thymocyte cell transformation, and that cells expressing the combination muthIL7R-hTLX3 is sufficient to trigger T-cell leukemia in vivo. Figure Disclosures No relevant conflicts of interest to declare.

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