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.

MicroRNA-126 attenuates cell apoptosis by targeting TRAF7 in acute myeloid leukemia cells

2018 ◽  
Vol 96 (6) ◽  
pp. 840-846 ◽  
Author(s):  
Qian Ding ◽  
Qing Wang ◽  
Yi Ren ◽  
Hong Qian Zhu ◽  
ZhuYun Huang
Keyword(s):  
Acute Myeloid Leukemia ◽  
Western Blot ◽  
Cell Lines ◽  
Cell Apoptosis ◽  
Myeloid Leukemia ◽  
Flow Cytometric Analysis ◽  
Tumor Necrosis Factor Receptor ◽  
Luciferase Assay ◽  
Acute Myeloid Leukemia Cells ◽  
Acute Myeloid

Acute myeloid leukemia (AML) has a 5-year survival rate of only about 30%–40% due to the self-renewal and differentiation ability of leukemia stem-like cells (LSCs). To address the potential for novel therapeutic targets in LSCs, we investigated the roles of miRNA-126 and tumor necrosis factor receptor-associated factor 7 (TRAF7) in AML. We used qRT-PCR and Western blot to investigate the expression levels of miRNA-126 and TRAF7 in AML cell lines. Then, we uncovered the effect of miRNA-126 on AML cell proliferation and apoptosis by MTT assay and flow cytometric analysis, respectively. Furthermore, dual-luciferase assay and Western blot were used to determine the target of miRNA-126 in AML and the potential mechanism by which cell apoptosis is suppressed by miRNA-126. We found that miRNA-126 was highly expressed in all of the AML cell lines, and that inhibition of miRNA-126 significantly induced cell death through apoptosis. The suppression of apoptosis in AML with high expression of miRNA-126 was caused by down-regulating TRAF7, which blocked the c-FLIP pathway. The role of miRNA-126 in AML makes it a potential therapeutic target to improve clinical outcomes for patients with AML.

Identification of Sensitizing Targets to the Hypomethylating Agent 5-Azacytidine in Acute Myeloid Leukemia Cells Using RNAi-Based Functional Genomics Screening

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2665-2665
Author(s):  
Raoul Tibes ◽  
Ashish Choudhary ◽  
Amanda Henrichs ◽  
Sadia Guled ◽  
Irma Monzon ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Growth Inhibition ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Statistical Significance ◽  
Molecular Targets ◽  
Leukemia Cells ◽  
Hypomethylating Agents ◽  
Acute Myeloid Leukemia Cells ◽  
Acute Myeloid

Abstract • Hypomethylating agents like 5-Azacytidine (5Aza) have become an effective therapy for myelodysplastic syndromes (MDS) and show promise in acute myeloid leukemia (AML). In AML, complimentary mechanisms including epigenetic silencing of growth controlling genes, i.e. tumor suppressors, and activation of kinases contribute to malignant transformation. In order to enhance the therapeutic potential of epigenetic therapies, we developed a high-throughput RNA interference (HT-RNAi) platform for large-scale transient gene silencing in acute myeloid leukemia cells. This assay allows for the first time to individually silence hundreds or thousands of genes in combination with 5Aza to identify molecular targets whose inhibition enhances the anti-leukemic effect of hypomethylating agents. As part of assay development for HT-RNAi, ten AML cell lines were used to determine the median inhibitory concentration (IC50) of 5Aza for each AML cell lines. Furthermore, the ten cell lines were tested with a panel of cationic lipid transfection reagents at varying weight to volume (wt:vol) ratios to determine the optimal siRNA transfection conditions. Results from these studies identified two AML cell lines TF1 and ML4, which were advanced into kinome-epigenetic RNAi screens. Using a lipid-based method, cells were reverse transfected for 48hrs with 2 different siRNA sequences per gene targeting a total of 572 kinases. After 48hrs, 5Aza at the calculated IC25 was added for an additional 72 hrs and cell proliferation was measured using a luminescence-based assay. Data was background corrected and analyzed using the B-score method to report the strength and statistical significance of growth inhibition compared to controls. A B-score of <−2 indicates statistical significance with p<0.05 (>95% confidence); a B-score <−1.5 provides >87% confidence and was used as lowest cutoff given that screens are focused and contain validated siRNA to kinases. Analysis of two independent RNAi kinome screens, one in TF1 and the other in ML4, in combination with 5Aza, identified six and eleven kinases respectively whose silencing by two different siRNA sequences (2× coverage) potentiated the effects of 5Aza at B-score <−1.5. In ML4 cells 2 kinases were highly significant with a B-score for both siRNA <−2. Six kinases were common targets in both cell lines with significant growth inhibition at a B-score for both siRNA of at least <−1.5 making these kinases potential important modifiers of response to 5Aza. In summary, initial kinome RNAi screens in myeloid cells identified specific kinases as potential sensitizing targets to hypomethylating agents. Moreover, functional genomic RNAi screens provide a fast and attractive approach to identify molecular targets in AML for the rational development of combination therapies with hypomethylating agents as well as other drug classes.

scholarly journals Discovery of putative tumor suppressors from CRISPR screens reveals rewired lipid metabolism in acute myeloid leukemia cells

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
W. Frank Lenoir ◽  
Micaela Morgado ◽  
Peter C. DeWeirdt ◽  
Megan McLaughlin ◽  
Audrey L. Griffith ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Saturated Fatty Acids ◽  
Leukemia Cell ◽  
Loss Of Function ◽  
Suppressor Activity ◽  
Acute Myeloid Leukemia Cells ◽  
Acute Myeloid

AbstractCRISPR knockout fitness screens in cancer cell lines reveal many genes whose loss of function causes cell death or loss of fitness or, more rarely, the opposite phenotype of faster proliferation. Here we demonstrate a systematic approach to identify these proliferation suppressors, which are highly enriched for tumor suppressor genes, and define a network of 145 such genes in 22 modules. One module contains several elements of the glycerolipid biosynthesis pathway and operates exclusively in a subset of acute myeloid leukemia cell lines. The proliferation suppressor activity of genes involved in the synthesis of saturated fatty acids, coupled with a more severe loss of fitness phenotype for genes in the desaturation pathway, suggests that these cells operate at the limit of their carrying capacity for saturated fatty acids, which we confirm biochemically. Overexpression of this module is associated with a survival advantage in juvenile leukemias, suggesting a clinically relevant subtype.

OncoHist®, an rh Histone 1.3, Is Cytotoxic to Acute Myeloid Leukemia Cells and Results in Altered Downstream Signaling

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3604-3604
Author(s):  
Suiyang Liu ◽  
Surender Kharbanda ◽  
Richard M. Stone
Keyword(s):  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Histone H1 ◽  
Chemotherapeutic Agents ◽  
P53 Mutation ◽  
Blue Staining ◽  
Downstream Signaling ◽  
Acute Myeloid Leukemia Cells ◽  
Acute Myeloid

Abstract Acute myeloid leukemia (AML) is a heterogeneous myeloid stem cell disorder; subtypes, which can be defined histologically, cytogenetically, and genetically, have varying prognoses and clinical characteristics. However, all but those younger patients with favorable biological features are inherently resistant to available cytotoxic chemotherapeutic agents; novel and less toxic therapies are required. Studies have shown that exposure of cancer cells, including leukemic blasts, to histone H1 produce cytotoxicity. The mechanism of cytotoxicity is believed to be an increase in focal membrane permeability induced by histone H1 binding to phosphotidyl serine moieties. However, the precise mechanism and functional effects of histone H1 on AML cells is currently unclear. The present study was aimed at investigating the effect of recombinant human Histone 1.3, OncoHist® (Xenetic Biosciences), on the proliferation of AML cell lines and on primary AML cells. We demonstrate that purified OncoHist® exerts growth inhibition and induces necrosis by flow cytometry assessment of staining for propidium iodide (PI) of AML cell lines (MOLM14, MV4-11, U937, HL60) as well as primary AML (n=3, including one with a p53 mutation) cells independent of lineage, stage and maturation with an IC50 of 2-5 mM. An AML cell line (MOLM13) with a FLT3 activating mutation (length or internal tandem duplication) which was developed to express resistance to the FLT3 inhibitor midostaurin/PKC412 was sensitive to OncoHist®-induced growth arrest measured by trypan blue staining and death (flow for PI) with about 70-80% necrosis at 5mM. Furthermore, 2uM OncoHist® treatment of MV4-11 and MOLM-14 cells for 15 minutes was associated with inhibition of the FLT3 downstream effectors phospho-AKT and phospho-extra cellular regulated kinase (phospho-ERK) as assessed by immunoblotting. Moreover, treatment of MOLM14 cells with OncoHist® in combination with cytarabine was associated with a significant synergistic inhibition of growth as measured by Alamar blue staining with a CI index of 0.35. Our findings support the development of OncoHist® alone and in combination with chemotherapy for the treatment of AML. A Phase I trial of OncoHist® for the treatment of refractory/relapsed AML is planned. Disclosures Kharbanda: Xenetic: Equity Ownership. Stone:Xenetic: Consultancy.

Autophagy Inhibition Overcomes Resistance to Cytosine Arabinoside in Acute Myeloid Leukemia Cells through Inducing Autophagic Cell Death and Intrinsic Pathway Apoptosis

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 909-909
Author(s):  
Yundeok Kim ◽  
Ju-In Eom ◽  
Hoi-Kyung Jeung ◽  
Jieun Jang ◽  
Jin Seok Kim ◽  
...  
Keyword(s):  
Cell Death ◽  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Cytosine Arabinoside ◽  
Myeloid Leukemia ◽  
Beclin 1 ◽  
Leukemia Cells ◽  
Intrinsic Pathway ◽  
Acute Myeloid Leukemia Cells ◽  
Acute Myeloid

Abstract Background A major obstacle to the successful treatment of acute myeloid leukemia (AML) is the development of chemoresistance. Identifying the novel agents overcoming drug resistance is critical for improving AML outcomes. Autophagy is an indispensable lysosomal self-digestion process involved in the degradation of aggregated proteins and damaged organelles. Autophagy has recently been demonstrated as important for conferring resistance to chemotherapy and targeted therapy. The antimalarial drug hydroxychloroquine (HCQ) is able to inhibit autophagy and therefore is being considered for cancer therapeutics. However, the effects of HCQ on chemoresistant myeloid leukemia cells have not been investigated. Objective The present study was designed to examine comparatively the effects of HCQ on the induction of cell death of the chemosensitive and chemoresistant acute myeloid leukemia cells, and elucidate its detailed mechanism. Method Ara-C (Cytosine arabinoside)-sensitive (U937, AML-2) and Ara-C-resistant (U937/AR, AML-2/AR) human myeloid leukemia cell lines were used to evaluate HCQ-induced cytotoxicity, autophagy, and apoptosis, as well as effects on cell death-related signaling pathways. Result U937/AR cell line showed a significantly higher number of autophagic vesicles and higher level of autophagic proteins. We initially found that HCQ caused dose- and time-dependent cell death of myeloid leukemia cells evaluated. HCQ-induced cell death rate was significantly higher in the chemoresistant U937/AR, AML-2/AR compared to chemosensitive U937 and AML-2 cells, respectively. Particularly, in Ara-C-resistant cell lines, HCQ triggers the activation of autophagy based on the results of increased number of autophagosomes, conversion of microtubule-associated protein light chain 3 (LC3)-I to LC3-II, and formation of GFP-LC3-positive punta. However, p62/SQSTM1 level was increased, suggesting that HCQ blocks the degradation of p62/SQSTM1 and autophagy flux. Modest upregulation of beclin-1 and Atg7 (autophagy-related protein 7) was observed. With continued exposure to HCQ, LC3 conversion was followed by nuclear condensation, procaspase-3 and -9 activation, release of cytosolic cytochrome C, and decreased mitochondrial membrane potential, indicating apoptosis via a mitochondria-dependent pathway. Pretreatment of leukemia cells with the autophagy blocker 3-methyladenine or siRNAs against beclin-1 or p62/SQSTM1, reduced HCQ-induced cell death, LC3 conversion, and procaspase-9 cleavage. The pan-caspase inhibitor z-VAD-fmk and the caspase-9 inhibitor z-LEHD-fmk, but not the caspase-8 inhibitor z-IETD-fmk, reduced HCQ-mediated cell death and caspase activation. However, LC3 conversion was unaffected. Additionally, Ara-C and HCQ synergistically induced cell death in U937/AR cells. Conclusion Taken together, our data show that HCQ effectively induced cell death in Ara-C-resistant AML cells through activation of autophagy and subsequent intrinsic pathway apoptosis. Our findings suggest HCQ might improve the therapeutic outcome in chemoresistant AML. Disclosures No relevant conflicts of interest to declare.

scholarly journals Inhibition of Autophagy Does Not Re-Sensitize Acute Myeloid Leukemia Cells Resistant to Cytarabine

2021 ◽  
Vol 22 (5) ◽  
pp. 2337
Author(s):  
Nienke Visser ◽  
Harm Jan Lourens ◽  
Gerwin Huls ◽  
Edwin Bremer ◽  
Valerie R. Wiersma
Keyword(s):  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
De Novo ◽  
Differential Sensitivity ◽  
Resistant Disease ◽  
Acute Myeloid Leukemia Cells ◽  
Autophagy Pathway ◽  
Arac Treatment ◽  
Acute Myeloid

Elevated activation of the autophagy pathway is currently thought to be one of the survival mechanisms allowing therapy-resistant cancer cells to escape elimination, including for cytarabine (AraC)-resistant acute myeloid leukemia (AML) patients. Consequently, the use of autophagy inhibitors such as chloroquine (CQ) is being explored for the re-sensitization of AraC-resistant cells. In our study, no difference in the activity of the autophagy pathway was detected when comparing AraC-Res AML cell lines to parental AraC-sensitive AML cell lines. Furthermore, treatment with autophagy inhibitors CQ, 3-Methyladenine (3-MA), and bafilomycin A1 (BafA1) did not re-sensitize AraC-Res AML cell lines to AraC treatment. However, in parental AraC-sensitive AML cells, treatment with AraC did activate autophagy and, correspondingly, combination of AraC with autophagy inhibitors strongly reduced cell viability. Notably, the combination of these drugs also yielded the highest level of cell death in a panel of patient-derived AML samples even though not being additive. Furthermore, there was no difference in the cytotoxic effect of autophagy inhibition during AraC treatment in matched de novo and relapse samples with differential sensitivity to AraC. Thus, inhibition of autophagy may improve AraC efficacy in AML patients, but does not seem warranted for the treatment of AML patients that have relapsed with AraC-resistant disease.

scholarly journals Apatinib Enhances Chemosensitivity of FLT3-ITD Mutations acute Myeloid Leukemia Cells to Homoharringtonine Via VEGFR2 Pathway

2021 ◽  
Author(s):  
Yuanfei Shi ◽  
Yan Zhang ◽  
Huafei Shen ◽  
Xiujin Ye ◽  
Jie Jin ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Growth Factor Receptor ◽  
Downstream Signaling ◽  
Acute Myeloid Leukemia Cells ◽  
Induced Apoptosis ◽  
Endothelial Growth Factor ◽  
Acute Myeloid

Abstract Purpose: The significance of vascular endothelial growth factor receptor (VEGFR)-3 in numerous solid tumors and acute myeloid leukemia (AML) has been demonstrated, but Apatinib remains largely unexplored. In this study, we explored whether Apatinib combined with HHT could kill AML cell lines and its possible mechanisms. Methods: All cell lines were treated with Apatinib and HHT in different concentrations with control, Apatinib alone, HHT alone, and Apatinib combined with HHT. The changes of IC50 were measured by CCK8 assay, apoptosis rate, cell cycle, and mitochondrial membrane potential in each group were measured by flow cytometry. Finally, the possible cytotoxicity mechanism was analyzed by Western blotting. Results: Our results noted that Apatinib combined with HHT remarkably inhibited cell proliferation, reduced the capacity of colony-forming, and induced apoptosis and cell cycle arrest in AML cells. Mechanistically, Apatinib and HHT play a role as a suppressor in the expression of VEGFR2 and the downstream signaling cascades, such as the PI3K, MAPK, and Stat3 pathways. Conclusion: Our preclinical data demonstrate that Apatinib combined with HHT exerts a better anti-leukemia effect than Apatinib alone by inhibiting the VEGFR2 signaling pathway, suggesting the potential role for Apatinib and HHT in the treatment of AML.

scholarly journals CD157 signaling promotes survival of acute myeloid leukemia cells and modulates sensitivity to cytarabine through regulation of anti-apoptotic Mcl-1

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yuliya Yakymiv ◽  
Stefania Augeri ◽  
Cristiano Bracci ◽  
Sara Marchisio ◽  
Semra Aydin ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Ex Vivo ◽  
Specific Inhibitor ◽  
Predictive Marker ◽  
Adp Ribosyl Cyclase ◽  
Acute Myeloid Leukemia Cells ◽  
The Impact ◽  
Acute Myeloid

AbstractCD157/BST-1 (a member of the ADP-ribosyl cyclase family) is expressed at variable levels in 97% of patients with acute myeloid leukemia (AML), and is currently under investigation as a target for antibody-based immunotherapy. We used peripheral blood and bone marrow samples from patients with AML to analyse the impact of CD157-directed antibodies in AML survival and in response to cytarabine (AraC) ex vivo. The study was extended to the U937, THP1 and OCI-AML3 AML cell lines of which we engineered CD157-low versions by shRNA knockdown. CD157-targeting antibodies enhanced survival, decreased apoptosis and reduced AraC toxicity in AML blasts and cell lines. CD157 signaling activated the PI3K/AKT/mTOR and MAPK/ERK pathways and increased expression of Mcl-1 and Bcl-XL anti-apoptotic proteins, while decreasing expression of Bax pro-apoptotic protein, thus preventing Caspase-3 activation. The primary CD157-mediated anti-apoptotic mechanism was Bak sequestration by Mcl-1. Indeed, the Mcl-1-specific inhibitor S63845 restored apoptosis by disrupting the interaction of Mcl-1 with Bim and Bak and significantly increased AraC toxicity in CD157-high but not in CD157-low AML cells. This study provides a new role for CD157 in AML cell survival, and indicates a potential role of CD157 as a predictive marker of response to therapies exploiting Mcl-1 pharmacological inhibition.

Effect of treatment with hedgehog inhibitor, PF-04449913, on leukemia-initiation potential in acute myeloid leukemia cells.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. e13520-e13520 ◽  
Author(s):  
Yosuke Minami ◽  
Nobuaki Fukushima ◽  
Tomoki Naoe
Keyword(s):  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Stromal Cells ◽  
Myeloid Leukemia ◽  
Combined Treatment ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Stem Cell Population ◽  
Acute Myeloid Leukemia Cells ◽  
Acute Myeloid

e13520 Background: Aberrant activation of the Hedgehog (Hh) signaling pathway is involved in a variety of cancers, and required for maintenance of the leukemic stem cell population in several experimental systems. PF-04449913 (PF) is a novel oral small molecule inhibitor that targets Smoothened (SMO) in the Hh pathway. Treatment with PF has shown promising results regarding safety, tolerability, and early signs of efficacy in a phase 1 study of hematologic malignancies including acute myeloid leukemia (AML) (Jamieson C, et al. ASH, 2011). Methods: We used AML cell lines and primary AML cells in order to elucidate mechanisms and biomarkers in PF treatment. We also used a co-culturing system with HS-5 stromal cells, and an immunodeficient NOD/SCID/IL2rγnull (NOG) mouse model serially xenotransplanted with primary AML cells to evaluate effects of PF on AML propagation. Results: In vivo-treatment with PF attenuated leukemia-initiation potential in acute myeloid leukemia cells through the serial transplantation system, while limiting reduction of tumor burden in the primary leukemia system. Ex vivo-treatment with PF inhibited proliferation and minimally induced cell death in leukemia cell lines and primary AML cells increased expression of the myeloid differentiation marker, CD11b. In addition, PF treatment down-regulated mRNAs encoding downstream effector GLIs in the canonical Hh pathway using RQ-PCR assays and decreased nuclear expression of GLI-2 using immunofluorescence assays. Moreover, combined treatment with PF abrogated resistance to Ara-C in MOLM-14 cells co-cultured with HS-5 stromal cells. We are also investigating biomarkers in these models including CD markers (such as CD44) as well as the toxicity for normal cord blood cells with PF treatment. Conclusions: These results imply that PF treatment can attenuate leukemia-initiation potential in acute myeloid leukemia cells and improve AML therapy through overcoming the resistance to chemotherapy in the bone marrow microenvironment.

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