Unique Effects of p53−/− Leukemic Cells On Mesenchymal Stromal Cell Gene Expression Profile in Vitro

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3468-3468
Author(s):  
Xiaoyang Ling ◽  
Ye Chen ◽  
Peter P. Ruvolo ◽  
Vivian Ruvolo ◽  
Zhiqiang Wang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Biology ◽  
Stromal Cell ◽  
Conflicts Of Interest ◽  
Leukemic Cells ◽  
Bone Marrow Niche ◽  
In Vivo Experiments ◽  
Cell Gene Expression

Abstract Abstract 3468 Mesenchymal stromal cells (MSC) participate in the generation of the microenvironmental bone marrow niche which protects normal and leukemic stem cells from injuries, including chemotherapy. MSC produce numerous factors that aid in this function; however, little is known about how leukemic cells affect MSC. In this study, paired murine AML cells, MLL/ENL/FIT3-ITD/p53−/− and MLL/ENL/FIT3-ITD/p53wt, originally derived from C57BL/6 mice (Zuber et al. Genes & Dev. 2009), were co-cultured with MSC from the same strain. After 48 hrs, MSC were isolated by FACS sorting using CD45−/PDGFr+ as markers. Total RNA was profiled on Illumina WG6 mouse whole-genome bead arrays by standard procedures. The significance analysis of microarrays (SAM) method identified 429 differentially-expressed genes (DEG) whose expression in MSC differed significantly (false discovery rate, 10%) in co-cultures with p53−/− (C78) vs. p53wt (C147) leukemic cells. Differences in these DEG were highly consistent in replicates (Figure 1). The results demonstrate that: 1) p53 status (p53−/− vs. p53wt) of AML cells affects GEP patterns in co-cultured MSC. Comparison of the GEP in MSC co-cultured with p53−/− (78) or p53wt (147) (Fig 1) identified the following 5 genes that showed the most significant differences (up- or down-regulated): up-regulated: WNT16, WNT5, IGFBp5, GCNT1, ATP1B1; down-regulated: NOS2, DCN, CCL7, CCL2, CAR9, CCL4. These were selected for qPCR validation, and the results confirmed the array data. In addition, immunohistochemical staining showed that WNT16 was up-regulated in MSC co-cultured with p53wt leukemic cells. In addition, CXCL5 was found up-regulated in MSC co-cultured with p53−/− leukemic cells. These results were consistent with the GEP data. 2) Leukemic cells alter MSC Signaling proteins in vitro: Western blotting showed that Stat3, Akt, PTEN, CXCL5 and HIF-1α were up- regulated in MSC co-cultured with p53−/− leukemic cells as compared to p53wt leukemic cells (48 hrs). Additional analyses showed that the downstream targets of HIF-1α, VEGFa and VEGFc, but not VEGFb, were up-regulated. Taken together, these results suggest that 1) leukemic cells with different p53 genetic background co-cultured with normal MSC have profoundly differential effects on GEP of normal MSC; 2) MSC co-cultured with p53−/− leukemic cells resulted in increased levels of onco-proteins such as Akt and HIF-1α when compared to MSC co-cultured with p53wt leukemic cells. Results suggest, for the first time, that the genetics of leukemic cells determines gene expression in co-cultured MSC. In vivo experiments are in progress to provide in vivo evidence for the existence of a novel model of leukemia-stroma interactions where the genetics of the tumor cell impacts stromal cell biology. Disclosures: No relevant conflicts of interest to declare.

Dysregulated Bone Wound Repair and Marrow Functions in Senescence Accelerated Mice (SAMP6),

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3415-3415
Author(s):  
Michael W. Epperly ◽  
Regina P. O'Sullivan ◽  
Shaonan Cao ◽  
Tracy M. Dixon ◽  
Julie P. Goff ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Biology ◽  
Wound Repair ◽  
Stromal Cell ◽  
Constitutive Expression ◽  
Marker Genes ◽  
Unexpected Finding ◽  
Semisolid Medium

Abstract Abstract 3415 Introduction: The Senescence Accelerated-Prone mouse (SAMP6) shows normal growth followed by rapid aging, development of osteopenia and shortened lifespan, compared with control R1 mice. The bone defect has been attributed to reduced osteoblast potential of marrow stromal cells. We compared in vivo repair of tibial bone wounds. We compared in vitro hematopoiesis, irradiation sensitivity, stromal cell biology, and osteoblastogenesis with SamP6 and R1 marrow. We tested the hypothesis that SAMP6 mice have poor healing of osseous wounds and that in vitro properties of SAMP6 marrow are abnormal. Methods: As a model for spontaneous bone repair, a 2-mm unicortical wound was made in each proximal tibia. At intervals, wound diameter was measured on radiographs. Long term bone marrow cultures (LTBMCs) were established from other SAMP6 and R1 mice to measure hematopoiesis and to establish marrow stromal cell lines (MSCLs) for analysis. MSCL gene expression and sensitivity to irradiation were measured; osteoblastogenic potential was determined by culture in osteoblastogenic medium (1% FBS, 10 nM dexamathasone, 5 mM b-glycerophosphate, 50 mg/mL ascorbate-2-phosphate) and gene expression analysis for osteoblast markers alkaline phosphatase (ALP), PTH-receptor-1 (PTHR1), and Osteocalcin (OC). Results: There was a faster rate of closure of the tibial wounds in R1 than in SAMP6 mice; at day 21, wounds in R1 mice were 47% the size of those in SAMP6 mice (p = 0.032). For the first 24 weeks, SAMP6 LTBMCs had significantly greater hematopoiesis than R1, shown by more cobblestone islands (sites of stem cell activity) and by more multilineage colonies from nonadherent cells transferred to semisolid medium. After 24 weeks, however, hematopoiesis ceased in SAMP6 cultures but continued in the SAMR1 cultures for 40 weeks. There was constitutive upregulation of TGF-b, an inhibitor of hematopoiesis, in SAMP6 MSCLs, compared with R1 cells. SAMP6 MSCLs were more sensitive to radiation (Do of 1.5 ± 0.1 Gy), compared with R1 (Do of 4.0 ± 0.4 Gy; p = 0.0080). There was unexpected constitutive expression of ALP, PTHR1, and OC in SAMP6 MSCLs, compared with R1. In osteoblastogenic medium, there was greater expression of osteoblast marker genes in the SAMP6 line compared to R1. Conclusion: SAMP6 showed delayed bone wound repair. Marrow from SAMP6 mice showed shortened in vitro hematopoiesis and greater radiation sensitivity, indicative of greater oxidative stress, but the unexpected finding of constitutive expression of osteoblast genes suggest that in vivo factors are more important than innate cellular defects in marrow to account for impaired bone healing. Acknowledgments: This project was supported by NIAID U191A168021-06 and NIA R21AG034254. Disclosures: No relevant conflicts of interest to declare.

Differentiation arrest and stromal cell-independent growth of murine erythroleukemia cells are associated with elevated expression of ets-related genes but not with mutation of p53

1993 ◽  
Vol 13 (9) ◽  
pp. 5582-5592
Author(s):  
R J Nibbs ◽  
K Itoh ◽  
W Ostertag ◽  
P R Harrison
Keyword(s):  
Stromal Cells ◽  
Stromal Cell ◽  
Gene Activation ◽  
Leukemic Cells ◽  
Term Survival ◽  
Elevated Expression ◽  
D Cells ◽  
Murine Erythroleukemia

The ELM erythroleukemia is novel in that long-term survival of leukemic cells in culture (ELM-D cells) is dependent on contact with a bone marrow-derived stromal feeder cell layer. However, a number of stroma-independent (ELM-I) mutants that vary in their ability to differentiate in vitro in response to erythropoietin and interleukin-3 have been derived. We have attempted to define the genetic changes responsible for these different phenotypes. At the p53 locus in the primary leukemic cells, one copy of the gene has been lost whereas the other contains an 18-bp depletion, implicating its mutation as an early step in the development of the leukemia. Changes in ets gene expression have also been found. The Fli-1 gene region is rearranged in the primary tumor because of the insertion of a retrovirus inserted upstream of one Fli-1 allele, but this does not result in Fli-1 gene activation in any of the ELM-D or ELM-I cell lines except one. It seems significant that this line is the only one to have lost the ability to differentiate in response to erythropoietin. In addition, up-regulation of erg is associated with stromal cell-independent growth, since all ELM-I mutants have moderate levels of erg mRNA, whereas only low or undetectable levels are found in primary leukemic cells in vivo or in ELM-D cells in vitro. This up-regulation of erg mRNA seems to be important for stromal cell-independent growth, since ELM-D cells show elevated expression of the erg gene after separation from stromal cells. This seems to be made permanent in ELM-I mutants, since they do not down-regulate erg mRNA when grown in contact with stromal cells. We therefore propose that ets family members regulate both the survival and differentiation of erythroid cells.

The Role of the Microenvironment for CLL Proliferation and Survival: Gene Expression Profiling of Leukemic Cells Derived from Blood, Bone Marrow and Lymph Nodes Reveals the B-Cell Receptor and NF κB as Dominant Signaling Pathways

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 356-356 ◽  
Author(s):  
Yair Herishanu ◽  
Berengere Vire ◽  
Delong Liu ◽  
Federica Gibellini ◽  
Gerald E Marti ◽  
...  
Keyword(s):  
Gene Expression ◽  
Tumor Cells ◽  
Expression Profiling ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Leukemic Cells ◽  
Activation Markers ◽  
Bcr Signaling

Abstract The host microenvironment is important for proliferation and survival of leukemic cells in chronic lymphocytic leukemia (CLL). Numerous molecules, signaling pathways and cell types have been reported to enhance CLL cell survival. To date, most reports on such interactions are derived from in-vitro studies, where each study focused on a specific ligand/receptor interaction or candidate pathway. Here, we adopted a more global approach to evaluate in-vivo effects of the microenvironment on leukemic cell biology. CLL cells from 15 patients were obtained on the same day from 3 different compartments: peripheral blood (PB), bone marrow (BM) and lymph node (LN), from which a single cell suspension was prepared. Tumor cells from all three compartments were purified by CD19 selection to purity >98%. Patients were assigned to prognostic subtypes based on immunoglobulin sequencing (Ig) and ZAP70 expression: 10 patients had the more progressive subtype (Ig-unmutated, ZAP70+) and 5 patients belonged to the more indolent subtype. Cells were analyzed for surface markers by flow cytometry and by gene expression profiling on Affymetrix HG U133 Plus 2.0 arrays. By flow cytometry, CLL cells in LN expressed higher levels of activation markers including CD69 and CD38 compared to CLL cells in PB (% CD19+/69+; 71 ±27 vs. 35 ±28, p<0.001 and % CD19+/CD38+; 33 ±28 vs. 20±19, p<0.001, respectively). The expression of activation markers in BM derived cells was less consistent and did not reach statistically significant differences. We therefore focused our analysis on a comparison between LN and PB derived cells. First, we confirmed that the expression of a diagnostic CLL gene expression signature established previously for PB derived cells (Klein et al, 2001) was equally present in leukemic cells derived from all three compartments. We then identified a set of about 275 genes that were differentially expressed between LN resident and circulating tumor cells, most of which were up-regulated (fold change >2, FDR <0.2). A large number of these genes encode proteins important for cell cycle control and proliferation: different cyclins, PCNA, Ki67, TOP2A and MYC. We also detected a significant increase in the expression of NF-κB target genes in LN resident tumor cells, including CD83, CD69, JunB, Cyclin D2, GADD45B, CCL3, CCL4 and others. Consistent with activation of the NF-κB pathway in LN, IκB-beta protein levels in tumor cells from LN were lower than levels in matching PB cells. Next we identified genes differentially expressed between CLL subtypes based on Ig-mutation status separately for each of the 3 compartments. Interestingly, these subtype identifying gene sets were only partially overlapping. In Ig-unmutated, ZAP70+ cells several genes were more strongly regulated by the microenvironment then in Ig-mutated, ZAP70 negative cells. Among these genes is LPL, which has been reported to distinguish the CLL subtypes, and other genes induced by B-cell receptor (BCR) signaling. Using in-vitro IgM activation, we show that these genes are indeed induced by BCR stimulation but not by CD40 ligation and that their induction is confined to ZAP70+ CLL cells. In conclusion: interactions between CLL cells and elements of the microenvironment in LN induce cell proliferation and NF-κB activation. The preferential upregulation of BCR regulated genes in ZAP70+ CLL demonstrates a more efficient in-vivo response of ZAP-70+ cells to BCR stimulation. Our results highlight the importance of NFκ κB and BCR signaling in CLL and provide a rationale to focus treatment approaches on these central pathways.

The Outgrowth of CLL Nurselike Cells in Vitro, and Its Relation to Clinical and Hematological Parameters.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2360-2360
Author(s):  
Agata A Filip ◽  
Dorota Koczkodaj ◽  
Tomasz Kubiatowski ◽  
Ewa Wasik-Szczepanek ◽  
Anna Dmoszynska
Keyword(s):  
Gene Expression ◽  
Serum Level ◽  
Ex Vivo ◽  
Hematological Parameters ◽  
Tp53 Gene ◽  
Leukemic Cells ◽  
Rt Pcr ◽  
Gene Status

Abstract Abstract 2360 Poster Board II-337 Introduction: Despite their longevity in vivo, CLL lymphocytes die rapidly when put to in vitro cultures, what proves that the resistance to apoptosis is not an intrinsic feature of leukemic cells, but depends on environmental signals. Recently it was shown that mononuclear cells from peripheral blood of CLL patients differentiate in vitro into large, adherent cells that grow in close contact with CLL lymphocytes. They were termed “nurselike cells” (NLCs), because they support leukemic lymphocyte survival in culture. The presence of the cells morphologically and phenotypically similar to NLCs was demonstrated in peripheral lymphatic organs of CLL patients. It may suggest their role in CLL lymphocytes protection in vivo and, as a consequence, point the new target in CLL treatment. Patients and Methods: The study included the group of 65 previously untreated CLL patients, 24 women and 41 men, aged from 36 to 86 yrs. 12 patients (18%) were diagnosed with stage 0 according to Rai, 15 patients (23%) with stage I, 30 patients (46%) with stage II, 5 patients (8%) with stage III and 3 patients (5%) with stage IV. Peripheral blood lymphocytes ex vivo were examined for CD14, CD38, BCL2 and ZAP70 expression by flow cytometry and for BCL2, SURVIVIN and ZAP70 gene expression by RT-PCR. TP53 gene status was assessed by FISH. Lymphocytes of 20 patients were assayed for apoptosis-related gene expression by means of cDNA macroarrays (Clontech). To generate NLCs, PB leukemic cells were cultured in vitro for 14 days on standard medium (RPMI 1640 with L-glutamine, 15% FCS, antibiotics/antimycotics; cell density 3 × 106/ml) and the outgrowth and number of NLCs was assessed in relation to clinical and hematological parameters. NLCs were identified morphologically and by CD31/VIMENTIN protein expression. Results: In 58 cases (89%) the outgrowth of NLCs was observed, while their number differed in cultures of the cells of different patients: in 49 cultures (84.5%) there were over 20 NLCs/mm2 (up to 52 NLCs/mm2), and in 9 cases (15.5%) less than 20 NLCs/mm2. Positive correlation was shown between NLC number and B2M serum level (p=0.044) and absolute monocyte count (p=0.019). Significantly higher NLC number was observed in case of patients with higher CD14+ cell number (p<0.0001) and higher SURVIVIN gene expression assessed by RT-PCR (p<0.0001) and macroarrays (p=0.013). We found no statistically significant relation of NLCs number and: the Rai stage of the disease, WBC, lymphocyte count, LDH serum level, BCL2, CD38 and ZAP70 expression and TP53 gene status. During the follow-up period of 6 years we observed the tendency for longer overall survival in patients that produce less than 20 NLCs/mm2 (fig. 1), but it was not statistically significant. Conclusions: The number of NLC cells obtained in vitro from PBL of CLL patients correlates with B2M serum level and SURVIVIN gene expression in CLL cells ex vivo. High B2M level is a marker of poor prognosis. SURVIVIN represents a family of IAP (Inhibitor of APoptosis) proteins. While rare in PBL of CLL patients, its expression is typical for proliferating leukemic cells pool in pseudofollicle microenvironment. SURVIVIN inhibits apoptosis by blocking caspase-3 and -7. Considering the protective role of NLC cells towards CLL lymphocytes in vitro, these results altogether with observed tendency to shorter survival of patients generating high NLCs number may prove the presence of supportive mechanisms exerted by NLCs in vivo. Disclosures: No relevant conflicts of interest to declare.

Identification of Non-Cardiotoxic hERG1 Blockers to Overcome Chemoresistance in Acute Lymphoblastic Leukemias

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1506-1506
Author(s):  
Marika Masselli ◽  
Serena Pillozzi ◽  
Massimo D'Amico ◽  
Luca Gasparoli ◽  
Olivia Crociani ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mouse Model ◽  
Map Kinases ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Significant Proportion ◽  
Leukemic Cells ◽  
K Channel

Abstract Abstract 1506 Although cure rates for children with acute lymphoblastic leukemia (ALL), the most common pediatric malignancy, have markedly improved over the last two decades, chemotherapy resistance remains a major obstacle to successful treatment in a significant proportion of patients (Pui CH et al. N Engl J Med., 360:2730–2741, 2009). Increasing evidence indicates that bone marrow mesenchymal cells (MSCs) contribute to generate drug resistance in leukemic cells (Konopleva M et al., Leukemia, 16:1713–1724, 2002). We contributed to this topic, describing a novel mechanism through which MSCs protect leukemic cells from chemotherapy (Pillozzi S. et al., Blood, 117:902–914, 2011.). This protection depends on the formation of a macromolecular membrane complex, on the plasma membrane of leukemic cells, the major players being i) the human ether-a-gò-gò-related gene 1 (hERG1) K+ channel, ii) the β1integrin subunit and iii) the SDF-1α receptor CXCR4. In leukemic blasts, the formation of this protein complex activates both the ERK 1/2 MAP kinases and the PI3K/Akt signalling pathways triggering antiapoptotic effects. hERG1 exerts a pivotal role in the complex, as clearly indicated by the effect of hERG1 inhibitors to abrogate MSCs protection against chemotherapeutic drugs. Indeed, E4031, a class III antiarrhythmic that specifically blocks hERG1, enhances the cytotoxicity of drugs commonly used to treat leukemia, both in vitro and in vivo. The latter was tested in a human ALL mouse model, consisting of NOD/SCID mice injected with REH cells, which are relatively resistant to corticosteroids. Mice were treated for 2 weeks with dexamethasone, E4031, or both. Treatment with dexamethasone and E4031 in combination nearly abolished bone marrow engraftment while producing marked apoptosis, and strongly reducing the proportion of leukemic cells in peripheral blood and leukemia infiltration of extramedullary sites. These effects were significantly superior to those obtained by treatment with either dexamethasone alone or E4031 alone. This model corroborated the idea that hERG1 blockers significantly increase the rate of leukemic cell apoptosis in bone marrow and reduced leukemic infiltration of peripheral organs. From a therapeutic viewpoint, to develop a pharmacological strategy based on hERG1 targeting we must consider to circumvent the side effects exerted by hERG1 blockers. Indeed, hERG1 blockers are known to retard the cardiac repolarization, thus lengthening the electrocardiographic QT interval, an effect that in some cases leads to life threatening ventricular arrhythmias (torsades de points). On the whole, it is mandatory to design and test non-cardiotoxic hERG1 blockers as a new strategy to overcome chemoresistance in ALL. On these bases, we tested compounds with potent anti-hERG1 effects, besides E4031, but devoid of cardiotoxicity (e.g. non-torsadogenic hERG1 blockers). Such compounds comprise erythromycin, sertindole and CD160130 (a newly developed drug by BlackSwanPharma GmbH, Leipzig, Germany). We found that such compounds exert a strong anti-leukemic activity both in vitro and in vivo, in the ALL mouse model described above. This is the first study describing the chemotherapeutic effects of non-torsadogenic hERG1 blockers in mouse models of human ALL. This work was supported by grants from the Associazione Genitori contro le Leucemie e Tumori Infantili Noi per Voi, Associazione Italiana per la Ricerca sul Cancro (AIRC) and Istituto Toscano Tumori. Disclosures: No relevant conflicts of interest to declare.

Inhibition of Casein Kinase 2 Impairs Wnt Signaling and Cell Survival in Chronic Lymphocytic Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2050-2050
Author(s):  
Christina Wu ◽  
Fitzgerald S Lao ◽  
Emily Nan ◽  
Hongying Li ◽  
Michael Y. Choi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Wnt Signaling ◽  
Conflicts Of Interest ◽  
Casein Kinase ◽  
Casein Kinase 2 ◽  
Lymphocytic Leukemia ◽  
Similar Reduction ◽  
Gene Assay

Abstract The oncogenic Wnt pathway is aberrantly activated in most CLL clones, and hence is an attractive target for therapy. The casein kinase 2 (CK2) enzyme is an established positive regulator of Wnt signaling. The inhibitor Silmitasertib, also known as CX-4945, is a nanomolar inhibitor of CK2. It has been reported that CK2 is overexpressed in CLL. Here we have investigated the effects of CX-4945 on WNT signaling in primary CLL cells. We confirmed that CX-4945 displayed in vitro cytotoxic activity toward CLL cells at very low µM concentration, as previously reported by others. However, at least 2-3 fold higher concentration of CX-4945 was required to achieve a similar toxicity against normal PBMC. Previously, our laboratory has successfully utilized a short-term CLL "parking" model in immunodeficient RAG/gamma chain knock out (RG-KO) mice to evaluate the in vivo efficacy and potential toxicity of anti-CLL agents. CX-4945 at dosages of 0.3-10 mg/kg was administered by oral gavage daily for 6 days to mice injected i.p. with 10 million CLL cells. These dosages of drug were well tolerated, and potently inhibited CLL persistence in the xenotransplanted mice. In a reporter gene assay, CX-4945 dose-dependently inhibited Wnt target gene expression. Furthermore, inhibition of dishevelled-2 (Dvl-2) protein expression was observed in primary CLL patient samples treated with 3-10 µM CX-4945 for 4-16 hours. Similar reduction in p-GSK3b(S9) protein was also observed. Quantitative RT-PCR also confirmed down regulation of b-catenin gene expression in primary CLL patient samples treated with 10 µM CX-4945 for 4h. Further molecular analyses of predictive or correlative biomarkers is ongoing using Nanostring PanCancer multipathway gene analysis. In a preliminary study, we found that CX-4945 perturbed the expression of multiple genes implicated in CLL development and survival. In summary, the CK2 inhibitor CX-4945 inhibited Wnt signaling and CLL survival, and displayed oral activity in mice. CK2 inhibitors are thus potential therapeutic agents for CLL. Disclosures No relevant conflicts of interest to declare.

scholarly journals Identification of Novel Lncrnas That Predict Survival in AML Patients and Modulate Leukemic Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3909-3909
Author(s):  
Sarah Grasedieck ◽  
Christoph Rueß ◽  
Nicole Pochert ◽  
Susanne Lux ◽  
Adrian Schwarzer ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Negative Impact ◽  
Strong Association ◽  
The Cancer Genome Atlas ◽  
High Expression ◽  
Leukemic Cells ◽  
Monocytic Differentiation ◽  
Granulocytic Differentiation

Abstract Long noncoding RNAs (lncRNAs) have complex, mainly chromatin-associated functions and their expression is highly coordinated and cell-type specific. Based on their tight regulation in normal differentiation, we set out to investigate whether lncRNAs are dysregulated in diseases where differentiation is impaired, such as in acute myeloid leukemia (AML). To identify lncRNAs that are essential for both normal hematopoiesis as well as AML maintenance, we sequenced the long polyA- and non-polyA-tagged transcriptome from successive stages of human myelopoiesis (myeloblasts, promyelocytes, metamyelocytes, and neutrophils) isolated from bone marrow of healthy donors (n=3). Applying a high-dimensional data portraying approach (OposSOM, Löffler-Wirth et al., BMC Bioinformatics, 2015), we identified functional expression modules of lncRNAs that are either positively or negatively associated with myeloid lineage commitment in our dataset. Seven out of the top15 differentiation-associated lncRNAs exhibit significant prognostic relevance in overall and event-free survival analyses of independent AML patient datasets and improve the predictive power of the current prognosis standards (cytogenetic risk/age/TP53-status). In particular, a combination of 3 transcripts, PROMYS (Promoter of Myelopoiesis, annotated as uncharacterized ncRNA LOC107985167), ANTAMY (Antagonist of Myelopoiesis, uncharacterized ncRNA LOC101927745) and LINC00677, outperformed the recently reported prognostic benefit of the LSC17high score (Ng et al, Nature, 2016) by a factor of Ø 22.7 based on concordance index score increase (Ø 4.8% vs. 0.21%). All three lncRNAs are highly conserved, expressed in 10 tested human AML cell lines as well as significantly differentially expressed in distinct cytogenetic patient subgroups of The Cancer Genome Atlas (TCGA) LAML cohort (n=171). PROMYS is downregulated in t(15;17) and t(8;21) cases, supporting its strong association with worse OS in the TCGA-LAML dataset (p=0.0001). In contrast, ANTAMY shows high expression in AML with t(8;21), and LINC00677 in NPM1+/FLT3- mutated AML patient samples with normal karyotype (CN-AML) and in core Binding factor (CBF) AMLs. Accordingly, high expression levels of both lncRNAs associate with a significantly better OS in the TCGA LAML dataset (p=0.01 and 0.02, respectively). To investigate their function in vitro, we knocked out each lncRNA individually in the human OCI/AML-5 AML cell line using CRISPR/Cas9. Loss of ANTAMY impaired proliferation (p=0.04) and increased both monocytic differentiation upon treatment with 2-0-tetradecanoylphorbol-13-acetate (TPA) (p=0.0001) and granulocytic differentiation with all-trans retinoic acid (ATRA) (p=0.0002) compared to the empty vector control. Loss of LINC00677 in OCI/AML-5 cells specifically increased granulocytic differentiation through ATRA (p=0.0002). In contrast, inactivation of PROMYS led to reduced differentiation induced by ATRA (p=0.00004) and TPA (p=0.002). Furthermore, we found that PROMYS is involved in the regulation of the Macrophage colony-stimulating factor 1 (CSF1), which is deregulated in ATRA- and TPA-induced differentiation in PROMYS knockout but not in control cells (p<0.002 and <0.00002, respectively), explaining its negative impact on differentiation. Through screening of human myelopoiesis, we identified three unexplored lncRNAs: LINC00677, PROMYS, and ANTAMY, which play a role in myeloid differentiation and have an impact on patient prognosis. Our in vitro findings confirm that ANTAMY, LINC00677, and PROMYS are active modulators of leukemic cells, which influence their proliferation, morphology, myeloid marker expression as well as apoptosis rate. These transcripts and their interaction partners add an additional layer of regulation to the understanding of differentiation and might represent previously unknown vulnerabilities of AML cells, which warrants their further investigation in vivo. Disclosures No relevant conflicts of interest to declare.

scholarly journals The autoregulation of a eukaryotic DNA transposon

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Corentin Claeys Bouuaert ◽  
Karen Lipkow ◽  
Steven S Andrews ◽  
Danxu Liu ◽  
Ronald Chalmers
Keyword(s):  
Binding Sites ◽  
Cell Biology ◽  
Eukaryotic Cell ◽  
Emergent Property ◽  
In Vivo Experiments ◽  
Constant Rate ◽  
Eukaryotic Dna ◽  
Transposase Expression

How do DNA transposons live in harmony with their hosts? Bacteria provide the only documented mechanisms for autoregulation, but these are incompatible with eukaryotic cell biology. Here we show that autoregulation of Hsmar1 operates during assembly of the transpososome and arises from the multimeric state of the transposase, mediated by a competition for binding sites. We explore the dynamics of a genomic invasion using a computer model, supported by in vitro and in vivo experiments, and show that amplification accelerates at first but then achieves a constant rate. The rate is proportional to the genome size and inversely proportional to transposase expression and its affinity for the transposon ends. Mariner transposons may therefore resist post-transcriptional silencing. Because regulation is an emergent property of the reaction it is resistant to selfish exploitation. The behavior of distantly related eukaryotic transposons is consistent with the same mechanism, which may therefore be widely applicable.

Sign in / Sign up

Export Citation Format

Share Document