Distinct Regulation of β- and γ-Catenin throughout Hematopoietic Development Contrasts with Their Cooperative Roles In Acute Myeloid Leukemia.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1573-1573
Author(s):  
Rhys Gareth Morgan ◽  
Lorna Pearn ◽  
Kate Liddiard ◽  
Robert Hills ◽  
Alan Burnett ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Wnt Signaling ◽  
Cell Lines ◽  
Nuclear Localization ◽  
Myeloid Leukemia ◽  
Nuclear Translocation ◽  
Differentiated Cells ◽  
Cd34 Cells ◽  
Normal Human ◽  
Acute Myeloid

Abstract Abstract 1573 Wnt proteins are important developmental regulators and are known to play a role in maintenance of hematopoietic stem cells (HSC). Wnt signaling has also been identified as one of the most frequently dysregulated processes associated with acute myeloid leukemia (AML), though the significance of this observation is as yet poorly understood. Here we investigate the role of two Wnt signaling proteins; β-catenin and γ-catenin and their respective roles in both normal human hematopoiesis and in AML. These proteins have dual and overlapping roles as transcriptional activators of Wnt target genes in the nucleus, and as structural components of the cytoskeleton. To determine the potential scope of influence of these proteins, we first examined their expression levels and subcellular location throughout normal human hematopoiesis using multi-parameter flow cytometric analysis and confocal microscopy. As expected β-catenin was strongly expressed in human cord blood derived HSC (212 MFI ±124, n=6) and at lower levels in differentiated subsets; surprisingly however β-catenin expression was maintained in granulocytic (1182 MFI±568) and monocytic cells (284 MFI±107). Nuclear localization was independent of cytoplasmic expression level, being strongly nuclear-localized in early progenitors and predominantly cytoplasmic in differentiated cells (58%±5 nuclear-localized in CD34+ cells vs 27%±1 in granulocytes, P=0.008). The expression pattern of γ-catenin was similar to β-catenin but showed a reciprocal pattern of subcellular localization, with levels of nuclear γ-catenin strongest in differentiated cells (10%±2 in CD34+ cells vs 44%±3 in monocytes P=0.0005). These data imply complementary roles for β and γ-catenin in normal hematopoiesis and show that nuclear localization of these proteins is regulated independently and irrespective of their expression level. In AML patients, β-catenin dysregulation has been previously reported; however, we also observed frequent overexpression of γ-catenin (over 5 fold in 25% of patients). This overexpression was associated with lower remission rates (OR 1.23 per log increase, P=0.03, CI 1.02–1.49) arising from resistant disease (OR 1.57 per log increase, P=0.003, CI 1.16–2.14) in a cohort of 243 AML patients adjusted for baseline diagnostic variables. In contrast to normal hematopoiesis, we found that nuclear localization of γ-catenin correlated with nuclear localization of β-catenin in AML (R=0.5, n=59) suggesting that the capacity to independently regulate the nuclear entry/retention of these catenins is disrupted in AML. To investigate this, we examined the effect of ectopic overexpression of γ-catenin in normal cord blood derived CD34+ cells and AML cell lines. Three-fold overexpression of γ-catenin failed to induce nuclear translocation of γ- or β-catenin in normal progenitors, which exhibited no major developmental defects. In contrast, in 3 of 4 AML cell lines, overexpression of γ-catenin strongly promoted its nuclear localization (9-16 fold) and was associated with a block in agonist-induced differentiation - a phenotype previously associated with β-catenin. In accord with this, we found that as in primary AML, nuclear translocation of γ-catenin in AML cell lines was associated with translocation of β-catenin (2-22 fold). In conclusion, we propose that in normal hematopoiesis, nuclear translocation of β- and γ-catenin is tightly and independently regulated for each catenin. In contrast, most AML cells lack this regulation resulting in correlated nuclear levels of β- and γ-catenin. In addition, we found while overexpression of γ-catenin has little consequence for normal cells; in malignant cells γ-catenin facilitates nuclear translocation of β-catenin. Disclosures: No relevant conflicts of interest to declare.

Triggering Noncycling Hematopoietic Progenitors and Leukemic Blasts to Proliferate Increases Anthracycline Retention and Toxicity by Downregulating Multidrug Resistance

Blood ◽  
1999 ◽  
Vol 94 (7) ◽  
pp. 2414-2423 ◽  
Author(s):  
Mariëlle E.P. Smeets ◽  
Reinier A.P. Raymakers ◽  
Gerty Vierwinden ◽  
Arie H.M. Pennings ◽  
Hans Wessels ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Multidrug Resistance ◽  
Myeloid Leukemia ◽  
Proliferation Rate ◽  
Related Protein ◽  
Hematopoietic Progenitors ◽  
P Glycoprotein ◽  
Cd34 Cells ◽  
Normal Human ◽  
Acute Myeloid

Expression of the multidrug resistance (MDR) mechanisms P-glycoprotein (Pgp) and MDR-related protein (MRP) decrease cellular retention and consequently cytotoxicity of anthracyclines. MDR is expressed on normal human hematopoietic progenitors and leukemic blasts. Normal CD34+ progenitors showed rhodamine efflux in 20% to 30% of the cells, which could be blocked by verapamil. These cells appeared noncycling, in contrast to the proliferating rhodamine bright (RhoB) cells. We postulated that MDR expression can be downregulated by proliferation induction. Triggering rhodamine dull (RhoD) CD34+ cells to proliferate indeed resulted in a higher rhodamine retention and significantly decreased efflux modulation by verapamil (P = .04). Also in acute myeloid leukemia (AML), the proliferation rate (percentage S/G2+M and Iododeoxyuridine labelings index) was significantly less in the RhoD blasts (P ≤ .008) and proliferation induction of RhoD blasts resulted in increased rhodamine retention. Anthracycline cytotoxicity was less for RhoD than RhoB cells in both normal progenitors and leukemic blasts. Proliferation induction of the RhoD cells resulted in increased anthracycline sensitivity. We conclude that noncycling progenitors, both normal and leukemic, have a relatively high MDR expression. Triggering these cells into proliferation downregulates MDR expression. These findings can be exploited to overcome MDR in the treatment of AML patients.

scholarly journals Differentiation-linked secretion of urokinase and tissue plasminogen activator by normal human hemopoietic cells.

1987 ◽  
Vol 165 (6) ◽  
pp. 1609-1623 ◽  
Author(s):  
E L Wilson ◽  
G E Francis
Keyword(s):  
Acute Myeloid Leukemia ◽  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Myeloid Leukemia ◽  
Leukemic Cells ◽  
Normal Cells ◽  
Differentiated Cells ◽  
Tissue Plasminogen ◽  
Normal Human ◽  
Acute Myeloid

Previous studies have shown that the response of patients with acute myeloid leukemia to induction chemotherapy can be predicted by the species of plasminogen activator that their cells secrete. Patients whose cells secreted tissue plasminogen activator (tPA) only failed to respond to combination chemotherapy. Individuals whose leukemic cells display features of the early progenitor phenotype also respond poorly to therapy. This suggested that the two species of plasminogen activator secreted by leukemic cells might be produced by normal cells at distinct stages of differentiation. These results indicate that the secretion of the two enzyme types is a differentiation-linked property of normal cells with tPA being produced by granulocyte/macrophage progenitors and urokinase by more differentiated cells and by mature neutrophils and macrophages.

scholarly journals FLT3 Is Sumoylated and Localizes to the Nucleus in Acute Myeloid Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2733-2733
Author(s):  
Marisse Asong ◽  
Julhash Uddin Kazi ◽  
Lars Ronnstrand
Keyword(s):  
Acute Myeloid Leukemia ◽  
Cell Surface ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Nuclear Translocation ◽  
Wild Type ◽  
Post Translational Modification ◽  
Subcellular Trafficking ◽  
Nuclear Entry ◽  
Acute Myeloid

FLT3 is a receptor tyrosine kinase (RTK) that is mutated and constitutively active in 30% of patients with acute myeloid leukemia (AML). Notably, mutations in FLT3 serve as poor prognostic indicator in AML patients. Despite its clinical relevance, the mechanisms of FLT3 subcellular trafficking remain poorly understood. Apart from the canonical downstream signaling cascades within the cytoplasm, accumulating data reveal direct nuclear translocation of RTKs. This alternative signaling pathway suggests that upon nuclear entry, RTKs can influence transcription and regulate gene expression. Interestingly, increased nuclear RTK levels have been linked to advanced tumor stages and poor clinical outcome. Whether or not the same phenomenon occurs in oncogenic FLT3 remains to be elucidated. This study focuses on establishing whether FLT3 undergoes nuclear translocation in AML. Moreover, we aim to understand the underlying subcellular trafficking mechanisms involved. First, the intracellular location of FLT3 was investigated in different human AML cell lines, stably transfected murine cell lines, as well as in primary patient samples. Subcellular fractionation protocols were optimized for each cell type and validated using protein markers for different cellular organelles. The cells were fractionated into cytoplasmic and nuclear fractions and analyzed by immunoblotting, immunofluorescent staining, and confocal imaging. Cells expressing FLT3 internal tandem duplication (FLT3-ITD) mutation show predominant cytoplasmic localization of FLT3. Interestingly, a basal level of FLT3-ITD was also detected in the nuclear fraction independent of ligand stimulation. To determine whether the same thing holds true for wild-type FLT3, AML cell lines which express wild-type FLT3 (FLT3-WT) were stimulated with its ligand for different time periods. FLT3-WT receptor was scarcely present in the nucleus under basal conditions. However, upon stimulation, a transient increase in nuclear FLT3 was observed. Using cell surface biotin labeling, we could verify that the detected nuclear FLT3 comes from the cell surface. Our results further show that nuclear FLT3 is phosphorylated to a certain degree, but that receptor activation is not required for nuclear translocation to occur. To investigate the subcellular trafficking mechanisms involved, the cells were treated with various small molecule inhibitors of cellular trafficking. Nuclear FLT3 levels were attenuated upon inhibition of clathrin and importin β, but stabilized by proteasomal inhibition. Modification of proteins by small ubiquitin-like modifier (SUMO) is a post-translational modification mainly observed in nuclear proteins. Since some RTKs require SUMOylation for nuclear entry, we investigated if this was also true for FLT3. Using immunoprecipitation, we could demonstrate that nuclear FLT3 gets SUMOylated by SUMO-1. To determine whether FLT3 SUMOylation is physiologically relevant, detection of endogenous SUMOylated FLT3 was done without overexpression of components of the SUMO machinery. Taken together, the present study demonstrates a previously uncharacterized post-translational modification and intracellular localization of FLT3. These data provide novel insights on the pathogenesis of AML and possibly new clues to improve targeted treatment of the disease. Additional in vitro studies and clinical data are necessary to establish the functional relevance of FLT3 translocation. On-going studies involve chromatin immunoprecipitation followed by sequencing (ChIP-seq) to gain further understanding on the biological function of nuclear FLT3. Ultimately, we will validate its clinical significance by analyzing nuclear FLT3 in AML patient samples and correlating this with patient outcome. Disclosures Ronnstrand: Acrivon Therapeutics: Consultancy.

Inhibition of IRAK1 As a Novel Therapeutic Strategy in Acute Myeloid Leukemia and Myelodysplastic Syndrome

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 612-612 ◽  
Author(s):  
Garrett Rhyasen ◽  
Lyndsey Bolanos ◽  
Jing Fang ◽  
Carmen Rigolino ◽  
Agostino Cortelezzi ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Protein Expression ◽  
Myelodysplastic Syndrome ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Kinase Activity ◽  
Myeloid Malignancies ◽  
Cd34 Cells ◽  
Acute Myeloid

Abstract Abstract 612 Recent work has shown that acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) patients exhibit downregulation of miR-146a, a miRNA that negatively regulates the innate immune pathway by targeting IRAK1 and TRAF6. Mice lacking miR-146a show elevated IRAK1 protein expression, and develop AML and MDS-like features resembling the human diseases. Prior to this study, the role of IRAK1 in human myeloid malignancies was unknown. We conducted a comparison of gene expression profiles of 136 cases of MDS CD34+ cells with 17 normal CD34+ cells obtained from ArrayExpress (E-GEOD-19429; Pellagatti et al., Leukemia, 2010). According to this data set, we observed IRAK1 overexpression in MDS patients (P = 0.017). IRAK1 is a serine/threonine kinase, and after phosphorylation on threonine-209 (T209), its kinase activity is induced, thus allowing for subsequent activation of TRAF6 and eventually NF-kB. Interestingly, we observed higher basal levels of phospho-IRAK1 at T209 in MDS and AML samples as compared to normal human CD34+ cells. To investigate the potential role of IRAK1 in AML and MDS, we used genetic and pharmacological approaches to suppress IRAK1 activity in MDS/AML cell lines and bone marrow cells from MDS patients. RNAi-mediated knockdown of IRAK1 in MDS and AML samples resulted in impaired growth of malignant hematopoietic stem/progenitor cells in methylcellulose assays and rapid apoptosis in vitro. In addition, we used a small-molecule inhibitor (benzimidazole analog; Amgen Inc.) to potently inhibit IRAK1 kinase activity. MDS/AML cell lines and MDS patient samples cultured with the IRAK1 inhibitor exhibited impaired growth and increased apoptosis, which coincided with decreased phospho-IRAK1 at T209, and active versions of TRAF6 and NF-kB. Importantly, the inhibition of IRAK1 kinase function is selectively detrimental to MDS and AML samples while preserving normal CD34+ cell viability and function. Given this novel requirement of IRAK1 in MDS and AML, we examined whether Lenalidomide or Bortezomib, two treatment options for MDS/AML and reported immunosuppressors, exhibit anti-leukemic activity in part by targeting IRAK1. We observed that Bortezomib, but not Lenalidomide, inhibits IRAK1 mRNA and protein expression in MDS/AML cells. The cytotoxic effect of Bortezomib can be partly rescued by forced expression of IRAK1 in these cells. To determine the molecular and cellular basis of cell death following loss of IRAK1 function or expression, we applied microarrays to MDS cells treated with IRAK1 inhibitor or transduced with a lentiviral vector encoding an shRNA targeting IRAK1. An overlap of commonly deregulated genes imposed by loss of IRAK1 expression or by the IRAK1 inhibitor revealed unique pathways relevant to the survival of MDS and AML cells. In summary, these findings are the first to implicate IRAK1 in the maintenance of myeloid malignancies and describe the effectiveness of an IRAK1 inhibitor on suppressing MDS and AML viability. Disclosures: Oliva: Celgene: Consultancy.

scholarly journals Acute Myeloid Leukemia with Mutated NPM1 Is Dependent on the Cytoplasmic Localization of NPM1c

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 877-877
Author(s):  
Lorenzo Brunetti ◽  
Michael Gundry ◽  
Anna Guzman ◽  
Ilaria Gionfriddo ◽  
Federica Mezzasoma ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Nuclear Localization ◽  
Myeloid Leukemia ◽  
Mutant Allele ◽  
Cytoplasmic Localization ◽  
Wild Type ◽  
Npm1 Mutation ◽  
C Terminus ◽  
Acute Myeloid

Abstract NPM1 mutations are among the most common mutations in acute myeloid leukemia (AML), being found in about 30% of de novo AML in adults. NPM1 is a multifunctional nucleolar chaperone involved in genomic stability and ribosome biogenesis. All the mutations in NPM1 described so far result in cytoplasmic protein localization (NPM1c) through the acquisition of a nuclear export signal (NES) at the C-terminus. Based on these observations, we hypothesized that cytoplasmic localization of NPM1c is necessary for AML pathogenesis and maintenance. To test our hypothesis, we edited the C-terminus of the NPM1 mutant allele in order to remove the NES and re-localize NPM1c to the nucleus. Specifically, using our recently optimized CRISPR-Cas9 approach (Gundry, et al. Cell Reports 2016), we sought to introduce indels to disrupt the C-terminal NES in AML cells bearing NPM1 mutation A (heterozygous 4bp insertion at the C-terminus), thereby creating novel edited alleles encoding for a mutant NPM1 with nuclear localization. Cell lines, primary derived xenografts (PDX) and primary AML samples harboring NPM1 mutation A were successfully edited with an sgRNA spanning the 4bp insertion. While the NPM1 wild-type allele remained intact, the mutant allele was edited with up to 90% efficiency. The novel edited alleles could direct nuclear localization of a GFP-NPM1 fusion construct. Additionally, re-localization of cytoplasmic NPM1 in edited cells was confirmed by immunofluorescence. Return of NPM1 protein to the nucleus resulted in terminal differentiation, and significantly impaired cell growth, colony forming ability and engraftment in xenograft models. Transcriptome analysis on two cell lines with mutated NPM1 revealed that upon nuclear re-localization of NPM1c, only 22 genes were down regulated more than 2 fold in both cell lines. Strikingly, 11 of these 22 genes were HOX genes. Altogether these data indicate that the cytoplasmic localization of NPM1c is necessary to maintain the transcriptional signature and leukemic phenotype of AML cells with mutated NPM1 . To extend our study further, we used homology-directed repair (HDR) to successfully correct NPM1 mutations to the wild type sequence in AML cell lines (Fig.1). We simultaneously fused GFP to the C-terminus of the protein to verify its appropriate localization (Fig.1). Moreover, to assess whether the phenotype of AML cells depends on the amount of mutant NPM1 in the cytoplasm, we inserted a series of NPM1 mutant alleles (a, b and c) that resulted in different proportions of cytoplasmic versus nuclear localization (i.e. a - no cytoplasmic mutant NPM1, b - ~50% cytoplasmic/50% nuclear mutant NPM1, c - ~100% cytoplasmic mutant NPM1; Fig.1) in two different AML cell lines with mutated NPM1 . Cells with high levels of nuclear NPM1 tended to differentiate rapidly, while cells with a greater proportion of cytoplasmic NPM1 remained less differentiated and continued to proliferate (Fig.1). This established an ongoing competition in which cells with higher nuclear NPM1 were rapidly outcompeted by unedited cells, while cells with higher cytoplasmic NPM1 were not. Thus, these experiments clearly demonstrate the dependence of the leukemic phenotype on the cytoplasmic localization of NPM1. Recently, compounds able to inhibit the nuclear exporter CRM1/XPO1 have been developed and are currently being tested in clinical trials (e.g. selinexor). Since NPM1 is shuttled to the cytoplasm by CRM1/XPO1, its inhibition re-localizes NPM1c to the nucleus. We therefore treated cell lines, PDX and primary samples with selinexor with the aim to reproduce the nuclear re-localization observed using CRISPR-Cas9. As expected, selinexor treatment resulted in clear growth arrest, differentiation, and NPM1c re-localization with dynamics similar to what was seen with gene editing. Importantly, after only 3 days of treatment selinexor produced a dramatic drop of HOXA, HOXB and MEIS1 expression. In conclusion, allele-specific editing is a powerful tool to probe the mechanistic aspects of oncogenic dependencies. By achieving nuclear re-localization of mutant NPM1, we demonstrated that cytoplasmic localization of NPM1c is necessary for NPM1-mutant AML cells to maintain their leukemic phenotype. Drugs promoting mutant NPM1 nuclear localization are therefore attractive candidates for clinical treatment of AML with mutated NPM1 . Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Antitumor Effect of Pomolic Acid in Acute Myeloid Leukemia Cells Involves Cell Death, Decreased Cell Growth and Topoisomerases Inhibition

2019 ◽  
Vol 18 (10) ◽  
pp. 1457-1468
Author(s):  
Michelle X.G. Pereira ◽  
Amanda S.O. Hammes ◽  
Flavia C. Vasconcelos ◽  
Aline R. Pozzo ◽  
Thaís H. Pereira ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Death ◽  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Natural Compound ◽  
Dna Topoisomerases ◽  
Human Dna ◽  
Pomolic Acid ◽  
Acute Myeloid

Background: Acute myeloid leukemia (AML) represents the largest number of annual deaths from hematologic malignancy. In the United States, it was estimated that 21.380 individuals would be diagnosed with AML and 49.5% of patients would die in 2017. Therefore, the search for novel compounds capable of increasing the overall survival rate to the treatment of AML cells is urgent. Objectives: To investigate the cytotoxicity effect of the natural compound pomolic acid (PA) and to explore the mechanism of action of PA in AML cell lines with different phenotypes. Methods: Three different AML cell lines, HL60, U937 and Kasumi-1 cells with different mechanisms of resistance were used to analyze the effect of PA on the cell cycle progression, on DNA intercalation and on human DNA topoisomerases (hTopo I and IIα) in vitro studies. Theoretical experiments of the inhibition of hTopo I and IIα were done to explore the binding modes of PA. Results: PA reduced cell viability, induced cell death, increased sub-G0/G1 accumulation and activated caspases pathway in all cell lines, altered the cell cycle distribution and inhibited the catalytic activity of both human DNA topoisomerases. Conclusion: Finally, this study showed that PA has powerful antitumor activity against AML cells, suggesting that this natural compound might be a potent antineoplastic agent to improve the treatment scheme of this neoplasm.

scholarly journals Chemotherapy-Induced Survivin Regulation in Acute Myeloid Leukemia Cells

2021 ◽  
Vol 11 (1) ◽  
pp. 460
Author(s):  
Petra Otevřelová ◽  
Barbora Brodská
Keyword(s):  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Survivin Expression ◽  
Mitotic Cell ◽  
Specific Subset ◽  
Suitable Target ◽  
Acute Myeloid Leukemia Cells ◽  
Almost All ◽  
Acute Myeloid

Survivin is a 16.5 kDa protein highly expressed in centrosomes, where it controls proper sister chromatid separation. In addition to its function in mitosis, survivin is also involved in apoptosis. Overexpression of survivin in many cancer types makes it a suitable target for cancer therapy. Western blotting and confocal microscopy were used to characterize the effect of chemotherapy on acute myeloid leukemia (AML) cells. We found enhanced survivin expression in a panel of AML cell lines treated with cytarabine (Ara-C), which is part of a first-line induction regimen for AML therapy. Simultaneously, Ara-C caused growth arrest and depletion of the mitotic cell fraction. Subsequently, the effect of a second component of standard therapy protocol, idarubicin, and of a known survivin inhibitor, YM-155, on cell viability and survivin expression and localization in AML cells was investigated. Idarubicin reversed Ara-C-induced survivin upregulation in the majority of AML cell lines. YM-155 caused survivin deregulation together with a viability decrease in cells resistant to idarubicin treatment, suggesting that YM-155 might be efficient in a specific subset of AML patients. Expression levels of other apoptosis-related proteins, in particular X-linked inhibitor of apoptosis (XIAP), Mcl-1, and p53, and of the cell-cycle inhibitor p21 considerably changed in almost all cases, confirming the off-target effects of YM-155.

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