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Jiang, Xuewei, Pan Zengkai, Chen Jin, Yu Pengfei, and Li-Gen Liu. "Establishment of TRAIL-Resistance Kasumi-1 Cell Line and the Analysis of It different mRNA Expression Profile with the Original Kasumi-1 Cell Line." Blood 124, no. 21 (December 6, 2014): 5221. http://dx.doi.org/10.1182/blood.v124.21.5221.5221.
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Abstract Introduction Tumor necrosis factor related apoptosis inducing ligand (TRAIL) can induce the apoptosis of many human leukemia cells while sparing of normal cells, but its resistance is also universal. Our previous study on apoptosis of t(8;21) positive acute myeloid leukemia cell line Kasumi-1 induced by rhTRAIL showed that the survival rate no longer decreased significantly when rsTRAIL reached a certain concentration which implied Kasumi-1 cells might have a resistant tendency to TRAIL. Then, we established a TRAIL-resistant Kasumi-1 cell line (Kasumi-1 TR) by intermittently escalating rsTRAIL concentration in culture media, and compared the mRNA expression profile with the original Kasumi-1 cell line by using Affymetrix Human Genome U133 Plus 2.0 Array. Methods Kasumi-1 TR cell line was established by intermittently treated Kasumi-1 cells with progressively escalating rsTRAIL concentration. Proliferation of leukemia cells were measured by CCK-8 assay, and rsTRAIL IC50 of cells and resistance index were calculated according to proliferation of cells treated with rsTRAIL at different concentrations. TRAIL and TRAIL receptors 1-4 on cells surface were detected by flow cytometry. Expression profiles of Kasumi-1 cells and Kasumi-1 TR cells were analyzed by Affymetrix Human Genome U133 Plus 2.0 Array to identify differentially expressed genes, and the search of genes possibly related with TRAIL-resistance were using by GO functional analysis and pathway enrichment analysis. Results 1) Kasumi-1 TR cells proliferation was faster than that of Kasumi-1 cells(Fig 1A); 2) IC50 of 24 hours for Kasumi-1 cells was 756.833ng/ml (logIC50 2.879 ± 0.148), IC50 of 24 hours for Kasumi-1 TR cells was 1634646.005ng/ ml (logIC50 6.213 ± 0.637), the RI of 24h was 2159 (Fig 1B); IC50 of 48 hours for Kasumi-1 cells was 345.390ng/ml (logIC50 2.538 ± 0.153), IC50 of 48 hours for Kasumi-1 TR cells was 33642.641ng/ml (logIC50 is 4.257 ± 0.317), the RI for 48h was 97 (Fig 1C); 3) Cell surface expression of TRAIL and its receptors 1-4 had no difference between two cell lines(Fig 1D). 4) There were 1537 genes up regulated by more than 2 times while 487 genes down regulated by more than 2 times in Kasumi-1 TR cells compared with the original Kasumi-1 cells (Fig 1E). Of which BCL-2 family antiapoptotic gene BCL2 is increased by 3.153 times and BCL2A1 increased by 18.23 times, IFNAR1 involved in JAK/STAT pathway increased by 12.841 times and TRAIL death receptor TNFRSF10A down regulated by 3.256 times(Fig 1F). Conclusions: The Kasumi-1 cell line with rsTRAIL resistance (Kasumi-1 TR) is established, and its resistance may be associated with the up expression of BCL2, BCL2A1, IFNAR1 and down regulated expression of DR4. Acknowledgment This work was supported by grants from NSFC (30672415) and STCSM (054119528). Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.
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平井一博. "A Study of「kasumi」「kasumu」after『The Tale of Genji』." Journal of Japanese Culture ll, no. 36 (February 2008): 211–23. http://dx.doi.org/10.21481/jbunka..36.200802.211.
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Imai, Norikazu, Masato Shikami, Hiroshi Miwa, Akiko Hattori, Akihito Hiramatsu, Masaya Watarai, Atsushi Satoh, et al. "Serum Dependency of t(8;21) AML Cell Line Is Associated with VEGF/VEGFR Pathway and Early Phosphorylation of Akt." Blood 106, no. 11 (November 16, 2005): 4571. http://dx.doi.org/10.1182/blood.v106.11.4571.4571.
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Abstract Most human leukemia cell lines are dependent on serum supplementation (usually fetal calf serum (FCS)), although the extent of serum dependency differs among each cell line. Kasumi-1, a t(8;21) AML cell line is one of the most serum-dependent cell lines. Since growth and survival of many leukemia cell lines are associated with phosphorylation of Akt, we examined the Akt phosphorylation by FCS treatment. In Kasumi-1, Akt was phosphorylated by culture with FCS in a dose-dependent manner, although no such Akt phosphorylation was observed in NB-4, a t(15;17) cell line. By FCS stimulation, Akt (Thr308, Ser473) was phosphorylated from 0.5 hr and the phosphorylation sustained until 48 hours in Kasumi-1. Then, we tested the effect of VEGF/VEGFR signaling in phosphorylation of Akt by FCS. The addition of VEGFR1/Fc and VEGFR2/Fc (which bind external VEGF and abrogate its function) inhibited the Akt phosphorylation from 2 hours until 10 hours, although the growth of Kasumi-1 was not inhibited. The addition of VEGFR2 kinase inhibitor (which inhibits internal VEGF signal) inhibited the Akt phosphorylation from 0.5 hr until 2 hours, and the growth of Kasumi-1 was greatly inhibited. Taken together, it is suggested that serum dependency of Kasumi-1 is at least in part attributed to VEGF/VEGFR pathway. Then, both external and internal VEGF/VEGFR pathways work in Kasumi-1, which in turn phosphorylate Akt. However, blockade of only internal VEGF signal (by VEGFR2 kinase inhibitor) inhibit the early Akt phosphorylation (0.5 hr), which resulted in growth inhibition, indicating the importance of early Akt phosphorylation.
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Kobune, Masayoshi, Kazuyuki Murase, Satoshi Iyama, Tsutomu Sato, Yutaka Kawano, Shohei Kikuchi, Koji Miyanishi, Rishu Takimoto, and Junji Kato. "Hedgehog Inhibitors Reduce the Survival and Drug Resistance in CD34+ Leukemic Cells." Blood 112, no. 11 (November 16, 2008): 1611. http://dx.doi.org/10.1182/blood.v112.11.1611.1611.
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Abstract Aberrant reactivation of Hedgehog (Hh) signaling has been described in a wide variety of human cancers and its role in maintenance of self-renewal of cancer stem cells. We previously showed that Indian Hh (Ihh) and its receptor molecules, Patched and Smoothened are expressed in cord blood (CB) CD34+ cells and Ihh regulate proliferation of hematopoietic myeloid- and lymphoid-repopulating cells in vivo. However, the involvement of Hh signaling in proliferation of leukemic cells has remained unclear. In this study, we assessed the possibility that Hh pathway activation contributes to the growth, survival and drug resistance of acute myeloid leukemic (AML) cells. Hh signaling in leukemic cell lines and bone marrow (BM) CD34+ cells were screened by RT-PCR and a Hh signaling reporter assay. We have found that Ihh were expressed in BM CD34+ cells and most of human AML cell lines, HL60, U937, KG1, Kasumi-1, Kasumi3 and TF-1. In contrast, Hh receptor components, Patched and Smoothened, were detected in BM CD34+ cells and cytokine responsible CD34+ AML cell lines such as Kasumi-1, Kasumi-3 and TF-1. Moreover, the downstream transcription factor GLI1 or GLI2 were expressed in BM CD34+ cells and these CD34+ cell lines, indicating that Hh signaling was active in these cells. We further assessed whether Hh signaling transmit to GLI1 using GLI1-responsive luciferase assay. GLI-responsive reporter plasmid (TK-6GBS-Luc) was transduced into these cells in the presence or absence of anti-Hh neutralizing antibody 5E1. TK-6GBS-Luc-transduced Kasumi-1, Kasumi-3 and TF-1 cells showed high luciferase activity. Furthermore, the luciferase activity of these cells was significantly reduced in the presence of 10 μg/ml anti-Hh neutralizing antibody. These results clearly indicate that Ihh signaling transmits into these CD34+ leukemic cell lines in autocrine manner. We next examined the effect of Hh inhibitors on these CD34+ leukemic cell lines. Inhibition of Hh signaling with the naturally derived chemical Smoothened antagonist Cyclopamine, endogenous Hh inhibitor Hedgehog-interacting protein or anti-Hedgehog neutralizing antibody reduces the entry of cell cycle of CD34+ cell lines and eventually induces apoptosis in these cells after 48 hr exposure although these CD34+ cell lines exhibit resistance to cytarabine (Ara-C) exposure. In contrast, cyclopamine did not affect growth and survival of the U937 or HL-60 cell line that lacks expression of Hh receptor components. Furthermore, combination of 10 μM cyclopamine significantly reduced the drug resistance against Ara-C in CD34+ cell lines. These results suggest that Hh pathway activation is a feature of CD34+ myeloid leukemic cells and inhibition of Hh signaling pathway may be a therapeutic strategy in the treatment of AML.
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Berg, Tobias, Mahmoud Abdelkarim, Yalin Guo, Manfred Fliegauf, and Michael Lübbert. "AML1/ETO Expresssion in Myeloid Leukemia Cells Is Associated with Enhanced Growth-Inhibitory and P15/INK4b Demethylating Effects of 5-AZA-2′-Deoxycytidine." Blood 104, no. 11 (November 16, 2004): 1165. http://dx.doi.org/10.1182/blood.v104.11.1165.1165.
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Abstract The chromosomal translocation (8;21) results in the expression of the chimeric transcription factor AML1/ETO, the most frequent fusion gene in acute myeloid leukemia. The AML1/ETO fusion protein acts as a transcriptional repressor by mediating epigenetic silencing through recruitment of histone deacetylases. Recently, it was shown that it also mediates gene silencing by associating with DNA methyltransferase (Dnmt). We therefore hypothesized that cells expressing AML1/ETO might be preferentially sensitive to the effects of an inhibitor of Dnmt activity, and might provide a superior model for in vitro demethylation and reactivation of the promoter of the p15/INK4b gene (encoding a negative regulator of the cell cycle) that is frequently methylated and silenced in AML and MDS. The 3 myeloid cell lines Kasumi-1 cells (AML1/ETO-positive), KG-1, and KG-1a (both AML1-ETO-negative) are all bearing a heavily methylated p15/INK4b promoter. They were treated with 50 – 1000 nM 5-aza-2′-deoxycytidine (DAC) for three pulses of 24 hrs each. After 6 days, cell growth and viability were determined and FACS analysis performed after propidium iodide staining. Kasumi-1 showed the highest sensitivity to DAC treatment (growth inhibition at 500 nM DAC: Kasumi-1 74.28 %, KG-1 69.16 %, KG-1a 62.38 %). In addition, DAC treatment (500 nM) led to a stronger increase in the sub-G1 fraction in Kasumi-1 (30.46%) compared to KG-1a (20.84 %). Regional p15/INK4b promoter methylation was assessed quantitatively by bisulfite sequencing of ≤10 individual cloned alleles (containing 21 CpGs residues) for calculation of methylated CpG percentage. The p15/INK4b was highly methylated in all 3 cell lines (methylated CpGs Kasumi-1 95.2 %; KG-1 89.6 %; KG-1a 98.4 % ). In Kasumi-1 cells, treatment with DAC resulted in a striking, dose-dependent regional demethylation of the p15/INK4b promoter (demethylated CpGs at 200 nM of DAC: Kasumi-1 63.8 %, KG-1 48.9 %, KG-1a 9.3 %). No demethylating effect was achieved with equitoxic doses of cytarabine or melphalan. Effective demethylation of the p15/INK4b promoter was associated with p15/INK4b protein induction as determined by Western Blot. Simultaneous treatment with all-trans retinoic acid (ATRA) enhanced the effects of DAC treatment upon growth inhibition, but not upon p15/INK4b induction. U937 cells with ecdysone inducible AML1/ETO expression (Fliegauf et al, Oncogene 2004) were also treated with different doses of DAC. When AML1/ETO was induced, U937 cells showed a higher growth inhibition (U937 + AML1/ETO 38.6 %, U937 - AML1/ETO 18 % at 25 nM) and increase in Sub-G1 (U937 + AML1/ETO 18 %, U937 - AML1/ETO 10.55 % at 100 nM) after treatment with DAC. Our results imply that the growth-inhibitory and proapoptotic effect of DAC on leukemia cells is modulated by AML1/ETO protein (or its target genes). The greater accessibility of the p15/INK4b promoter to the demethylating effect of DAC in AML1/ETO expressing Kasumi-1 cells may also be due to differences in regional chromatin structure. With their differential sensitivity to DAC, the cell lines Kasumi-1 and KG-1a provide a model for the different responses of leukemic blasts to DAC.
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KITSOS, P., M. D. GALANIS, and O. KOUFOPAVLOU. "AN FPGA IMPLEMENTATION OF THE GPRS ENCRYPTION ALGORITHM 3 (GEA3)." Journal of Circuits, Systems and Computers 14, no. 02 (April 2005): 217–31. http://dx.doi.org/10.1142/s0218126605002337.
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The General Packet Radio Service (GPRS) uses the GPRS Encryption Algorithm 3 (GEA3) for data encryption. In this paper, alternative hardware implementations of the GEA3 algorithm are described. GEA3 algorithm is based on the KASUMI block cipher. Various KASUMI block cipher hardware implementations have been examined in order to provide information about the required silicon area and throughput. In order to achieve a significant performance improvement, Double Edge Triggered pipeline technique is used. The S-BOXes, which are fundamental elements of the KASUMI cipher, have been implemented by using combinational logic and ROM memories. The proposed GEA3 algorithm hardware implementation achieves throughput up to 837Mbps, which is much faster comparing to the previous designs. The whole system is implemented and evaluated by using Field Programmable Gate Array (FPGA) devices.
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O'Connor, Christine M., and Eain A. Murphy. "A Myeloid Progenitor Cell Line Capable of Supporting Human Cytomegalovirus Latency and Reactivation, Resulting in Infectious Progeny." Journal of Virology 86, no. 18 (July 3, 2012): 9854–65. http://dx.doi.org/10.1128/jvi.01278-12.
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Human cytomegalovirus (HCMV) is a herpesvirus that establishes a lifelong, latent infection within a host. At times when the immune system is compromised, the virus undergoes a lytic reactivation producing infectious progeny. The identification and understanding of the biological mechanisms underlying HCMV latency and reactivation are not completely defined. To this end, we have developed a tractablein vitromodel system to investigate these phases of viral infection using a clonal population of myeloid progenitor cells (Kasumi-3 cells). Infection of these cells results in maintenance of the viral genome with restricted viral RNA expression that is reversed with the addition of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA, also known as PMA). Additionally, a latent viral transcript (LUNA) is expressed at times where viral lytic transcription is suppressed. Infected Kasumi-3 cells initiate production of infectious virus following TPA treatment, which requires cell-to-cell contact for efficient transfer of virus to other cell types. Importantly, lytically infected fibroblast, endothelial, or epithelial cells can transfer virus to Kasumi-3 cells, which fail to initiate lytic replication until stimulated with TPA. Finally, inflammatory cytokines, in addition to the pharmacological agent TPA, are sufficient for transcription of immediate-early (IE) genes following latent infection. Taken together, our findings argue that the Kasumi-3 cell line is a tractablein vitromodel system with which to study HCMV latency and reactivation.
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Sun, Jin, Shujun Liu, Jianhua Yu, Min Wei, Charlene Mao, Haiming Ding, Jessica Kearney, et al. "Characterization of HDACI OSU42 as a Novel Histone Deacetylase Inhibitor in AML Cell Lines." Blood 108, no. 11 (November 16, 2006): 1988. http://dx.doi.org/10.1182/blood.v108.11.1988.1988.
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Abstract Histone acetylation plays a key role in the regulation of gene expression. Histone hyperacetylation is associated with chromatin opening and gene transcription, while histone hypoacetylation is associated with chromatin condensation and gene silencing. Abnormal histone hypoacetylation mediated by aberrant activity of histone deacetylases (HDACs) has been found to be associated with silencing of tumor suppressor and growth inhibitory genes in malignant cells. HDAC inhibitors (HDACIs) can relieve HDAC-mediated gene silencing and thereby induce normal patterns of cell cycle, differentiation and apoptosis in malignant cells. HDACI OSU 42 is a novel hydroxamate tethered phenylbutyrate derivative that was designed and synthesized at our institution, and exhibited IC50s at submicromolar level, compared with millimolar level for other members of this classes of HDACIs such as valproic acid (VPA). We characterized the activity of this compound in acute myeloid leukemia (AML) cells. It is known that the fusion proteins AML1/ETO and PML / RAR alpha that characterized t(8;21) and t(15;17) AML silence target genes through recruitment of HDACs to their promoter regions. Therefore we utilized AML1/ETO-positive Kasumi-1 and PML/RARA-positive NB4 cells to test the activity of HDACI OSU 42 and used THP-1 cells, characterized by AF9/MLL fusion gene, as a control. We hypothesized that by virtue of the fusion genes, Kasumi-1 and NB4 are more susceptible to HDACI treatment. IC50s for proliferation inhibition in Kasumi-1 cells treated with HDACI OSU42 were 71.8±14.3nM for 24hr and 31.3± 0.4nM for 48hr, significantly lower than VPA (2.0mM for 24hr, 0.9mM for 48hr). The IC50s for NB4 were 237.7±6.5nM for 24hr and 119±6.4nM for 48hr. As a contrast, IC50 for THP-1 was 507.3±68.3nM for 48hr. HDACI OSU42 inhibited 80% of total HDAC activity at 125nM in both Kasumi-1 and NB4; 30nM HDACI OSU42 induced hyperacetylation of histone H3 and H4. Apoptosis analysis showed that nearly 60% more of Kasumi-1 and NB4 underwent apoptosis after treated with 1μM of HDACI OSU42 for 24hr, compared with their untreated control. On the other hand, the same treatment only induced 15% more of THP-1 undergoing apoptosis. Apoptotic effect of HDACI OSU42 was mediated by activation of caspase 9 and caspase 3. Cell cycle analysis demonstrated that treatment of Kasumi-1 and NB4 with 150nM of HDACI OSU 42 inhibited cell cycle progression and arrested 20% to 30% more cells at S phase or G2/M phase, whereas this treatment had not effect on cell cycle progression of THP-1. This was consistent with the up-regulated expression of p21 at both transcription level and protein level. Q-PCR data suggested that Kasumi-1 and NB4 treated with HDACI OSU42 expressed ~10 folds of p21 higher than untreated cells. Chromatin immunoprecipitation assay revealed 10 to 50 folds increase in acetylation level of histone H3 and H4 associated with p21 promoter. Kasumi-1 and NB4 cells also show differentiation ability (increase in CD14 and CD 13 expression by flow cytometry) when treated with 30nM of HDACI OSU42, whereas THP-1 remained undifferentiated. These results support the activity of HDACI OSU42 as a new potent HDACI in AML.
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Mahmud, Hasan, Frank JG Scherpen, Tiny Meeuwsen-de Boer, Harm-Jan Lourens, and Eveline S. de Bont. "Essential Role for Phospholipase C Gamma 1 (PLC-γ1) in the Survival of t(8;21) Acute Myeloid Leukemia." Blood 128, no. 22 (December 2, 2016): 1699. http://dx.doi.org/10.1182/blood.v128.22.1699.1699.
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Abstract The t(8;21) (q22;q22) chromosomal translocation is one of the most frequent genetic alterations in acute myeloid leukemia (AML) and it remains a significant clinical problem especially for children which indicates the need for improved therapeutic strategies. Recently, we showed that peptide derived phospholipase C gamma 1 (PLC-γ1) was highly phosphorylated in pediatric t(8;21) AML. In this study, we determined PLC-γ1 phosphorylation and mRNA levels showing that PLC-γ1 expression was significantly higher in t(8;21) AML compared to other AML karyotypes and normal bone marrow (NBM) (peptide phosphorylation: p<0.01 compared to NBM, mRNA: p<0.001, compared to other AML karyotypes). This was confirmed by PLC-γ1 protein phosphorylation using primary AML samples and AML cell lines. PLC-γ1 is known to play a role in cancer progression, however, the impact of PLC-γ1 in AML is currently unknown. Therefore, we aimed to study the functional role of PLC-γ1 by investigating the cellular growth, survival and its underlying mechanism in a t(8;21) AML cell line (Kasumi-1) . ShRNA-mediated knockdown of PLC-γ1 in kasumi-1 cells significantly blocked leukemic cell growth at day 8 after transduction (p<0.05). The percentage of apoptosis in PLC-γ1 suppressed kasumi-1 cells at day 4 after transduction was two-fold higher compared to the scrambled control (p<0.01). The inhibition of cell proliferation and the induction of apoptosis upon PLC-γ1 suppression could be explained by cell cycle arrest and by increased activation of apoptotic related and cell cycle regulatory protein expressions (BAX, BCL2, p53 and Chk2). As the multidrug resistance is one of the major cause of relapse and poor prognosis in t(8;21) AML, therefore, we demonstrated, if PLC-γ1 suppression increased the sensitivity of kasumi-1 leukemia cells to cytotoxic chemotherapeutic agents (methotrexate, amsacrine and etoposide). PLC-γ1 knockdown cells at day 4 after transduction were shown to significantly reduced cell viability to the genotoxic agents, methotrexate (p<0.05, p<0.001), amsacrine (p<0.01, p<0.001) and etoposide (p<0.05, p<0.01 and p<0.001) in kasumi-1 in a dose-dependent manner. These results provide a strong rationale for the development of PLC-γ1-based therapeutic strategies for the enhancement of efficacy in t(8;21) AML treatment. Additionally, PLC-γ1 suppressed kasumi-1 cells showed significantly less proliferation upon hypoxic stress. Taken together, these results strongly support an important role for PLC-γ1 in the survival of t(8;21) AML mimicking kasumi-1 cell line and identify PLC-γ1 as a potential target for t(8;21) AML treatment. Disclosures No relevant conflicts of interest to declare.
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Liu, Shujun, Jiuxia Pang, Jianhua Yu, Zhongfa Liu, Lenguyen Huynh, Jessica Kearney, Peter Paschka, et al. "Bortezomib-Induced Down-Regulation of KIT Is Mediated by Inhibition of Sp1 and NF-kB in AML1/ETO-Positive Cells." Blood 108, no. 11 (November 16, 2006): 4211. http://dx.doi.org/10.1182/blood.v108.11.4211.4211.
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Abstract Activating mutations of KIT, encoding a type III receptor tyrosine kinase, are frequently detected in core binding factor AML (i.e., AML1/ETO and CBFB/MYH11 AML), promote cell survival and proliferation of leukemic cells and predict poor outcome. Kinase inhibitors (e.g., imatinib or PKC-412) have been shown to block constitutively activated KIT. However, novel therapeutic approaches that target mutated KIT are necessary, since resistance to these agents can be predicted in a substantial proportion of patients in these subgroups of AML. We observed that expression levels of KIT in AML1/ETO-positive Kasumi-1 cells are more than 25 fold higher than in AML1/ETO-negative AML cell lines (i.e., THP-1, K562, and MV4–11). We also found that in Kasumi-1 cells, bortezomib (Velcade), a proteasome inhibitor already used in the clinic, induces time- (60nM for 0, 1, 3, 6 12 and 24hr) and dose- (0, 1, 6, 20, 60 and 100nM for 24hr) dependent down-regulation (>90%) of total KIT expression. Noticeably, dephosphorylation of KIT occurred 6hr before reduction of the total KIT level after bortezomib exposure. We also found >50% down-regulation of KIT expression in patients’ primary blasts treated with 60nM bortezomib for 24hr and in Kasumi-1 cells treated with the proteasome inhibitor MG132 (300nM for 24hr). Down-regulation of KIT appeared to be associated with inhibition of NF-kB and Sp1, which is necessary for regulation of the KIT promoter activity by the SCL complex. In fact, treatment with bortezomib inactivated NF-kB and decreased transcription of Sp1 in Kasumi-1 cells. Furthermore, exposure to the NF-kB or Sp1 inhibitors parthenolide (30μM for 24hr) and mithramycin (100ng/ml for 24hr), respectively, resulted in a dose-dependent decrease in KIT expression in Kasumi-1 cells. When cells were treated with bortezomib (20nM) in combination with mithramycin (30ng/ml for 24hr), we observed synergy in down-regulation of KIT RNA and KIT protein. This correlated with growth arrest and increased cell death. Interestingly, the magnitude of these effects was higher when Kasumi-1 cells were pretreated with mithramycin for 24hr before being exposed to bortezomib. Taken all together, our data suggest that bortezomib downregulates KIT expression and might also inhibit KIT phosphorylation and should be considered in future therapeutic strategies targeting AML subgroups harboring mutated KIT.
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Drescher, Bettina, Kerstin Goerlich, Dagmar Reile, Gerhard Heil, Olaf Heidenreich, Katharina Wagner, Arnold Ganser, and Juergen Krauter. "Decrease of Malignant Potential of t(8;21) Positive Cells after Stable Expression of RUNX-CBFA2T1-Specific Small Interfering RNA." Blood 104, no. 11 (November 16, 2004): 4274. http://dx.doi.org/10.1182/blood.v104.11.4274.4274.
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Abstract The reciprocal chromosomal translocation t(8;21) results in the fusion of the DNA-binding domain of the transcription factor RUNX1 (also called AML1 or CBFα) to CBFA2T1 (also called MTG8 or ETO). The chimeric protein RUNX1-CBFA2T1 interferes with hematopoietic gene expression by recruiting histone deacetylases via N-CoR and mSin3. In addition, impairment of the function of transcription factors such as C/EBPα has been described. To further investigate the function of RUNX1-CBFA2T1 in the development of leukemia, the expression of the fusion protein was inhibited by small interfering RNAs (siRNAs). For stable expression of the siRNA in the target cells, a RUNX1-CBFA2T1-specific siRNA-sequence was cloned into a lentivial vector as short-hairpin-RNA (shRNA). Subsequently, the t(8;21) positive Kasumi-1 cell line was infected with an effeciency of >95% as detected by expression of the GFP reporter gene. As a control, an shRNA targeting luciferase mRNA was used. Expression of the anti-RUNX1-CBFA2T1-shRNA in the Kasumi-1 cells led to a marked reduction of mRNA and protein expression of the fusion gene, whereas the expression of the wildtype RUNX1 gene was not affected. The surface expression of the M-CSF-Receptor, which is a known target gene of C/EBPα, was increased in the RUNX1-CBF2T1 depleted cells. Also, Kasumi-1 cells treated with the shRNA displayed a decrease in CD34 surface expression. In parallel, CD34 mRNA expression was reduced to 10%. To analyze, if CD34 downregulation of the Kasumi-1 cells after RUNX1-CBF2T1 depletion correlates with a loss of progenitor status, the clonogenicity of the cells in semisolid medium was investigated. In Kasumi-1 cells treated with the shRNA against RUNX1-CBFA2T1 the number of spontaneous colonies after 14 days of culture was reduced to about 30% in comparison to cells expressing the control shRNA. In conclusion, these experiments show that RUNX1-CBF2T1 expression can be stably suppressed in t(8;21) positive cells by endogenously expressed shRNAs thereby reducing their clonogenic potential.
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Tsai, Hui-Jen, Tsu-An John Hsu, Chiung-Tong Chen, Weir-Torn Jiang, Hui-You Lin, Sheng-Fung Lin, and Li-Tzong Chen. "1J373 Is a Promising Agent Against C-KIT driven Acute Myeloid Leukemia." Blood 122, no. 21 (November 15, 2013): 1448. http://dx.doi.org/10.1182/blood.v122.21.1448.1448.
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Abstract Acute myeloid leukemia (AML) carrying t(8;21)(q22;q22) and inv(16)/t(16;16)(p13;q22) are classified as French-American-British (FAB) AML subtype M2 or monocytic with eosinophilic differentiation (M4Eo) by morphology and as core binding factor (CBF)-AML according to pathogenesis. CBF-AML accounts for approximately 15% of AML and frequently harbors c-KIT mutation (17∼46%). C-KIT mutated CBF-AML patients usually have higher baseline white blood cell count, higher relapse rate and shorter event free survival/overall survival after conventional chemotherapy than those without c-KIT mutation. It is conceived that c-KIT mutation is a crucial hit and cooperates with AML1-ETO resulting from t(8;21)(q22;q22) to cause overt AML. Imatinib, a tyrosine kinase inhibitor suppressing c-KIT activation, has been used in c-KIT mutated AML, systemic mastocytosis (SM) and gastrointestinal stromal tumor (GIST). However, c-KIT exon17 D816V mutation, a frequent mutation of CBF-AML and SM, is associated with primary imatinib resistance. 1J373, a multi-targeted tyrosine kinase inhibitor, which was initially designed as a FLT3 inhibitor but later found to target c-KIT as well. It has been shown to effectively inhibit the proliferation of FLT3-ITD mutated leukemia cell lines, MV4;11 and MOLM-13, both in vitro and in vivo. (unpublished data) Among a series of myeloid leukemia cell lines without FLT3-ITD mutation, including THP-1, HL-60, K562, KG-1, and kasumi-1, the sensitivity to 1J373 is closely associated with the presence of constitutive c-KIT activation (Figure 1A). The IC50 of 1J373 for cells with (K562, KG-1 and kasumi-1) and without (THP-1 and HL-60) constitutive c-KIT activation was below 50 nM and beyond 1000 nM, respectively. 1J373 suppressed the phosphorylation of c-KIT for cell lines with constitutively activated c-KIT, which suggested that 1J373 may suppress the proliferation of KG-1, K562, and kasumi-1 by inhibiting c-KIT (Figure 1B).We further compared the efficacy of 1J373 and imatinib in kasumi-1, a cell line with t(8;21)/AML1-ETO and c-KIT exon 17 N822K mutation. At 1000nM of concentration, the phosphorylation of c-KIT was effectively inhibited by imatinib at 2-hour but partially recovered after 8 hours; while 1J373 treatment resulted in a sustained inhibition for 24 hours. The inhibition of c-KIT activation by both agents was accompanied with corresponding changes in the phosphorylation status of its downstream signaling pathway molecules, including PI3K, AKT, mTOR, and MAPK (Figure 2). 1J373 induced cell cycle arrest of kasumi-1 at G1 phase with increase of subG1 population time-dependently and induced apoptosis of kasumi-1 through activation of caspase 8 and 9, and upregulation of proapoptotic proteins Bax and Bak. The in vivo experiments are in progress. In conclusion, 1J373, a multi-targeted tyrosine kinase inhibitor, can effectively inhibit the proliferation and induce the apoptosis of c-KIT activated leukemia cells. It has the potential to be used in clinical practice to treat c-KIT driven, particularly c-KIT mutated, AML.Figure 1c-KIT and phosphorylated c-KIT expression in myeloid leukemia cell linesFigure 1. c-KIT and phosphorylated c-KIT expression in myeloid leukemia cell linesFigure 2c-KIT and its downstream signalings expression in kasumi-1 cells treated with imatinib and 1J373Figure 2. c-KIT and its downstream signalings expression in kasumi-1 cells treated with imatinib and 1J373 Disclosures: No relevant conflicts of interest to declare.
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Miwa, Hiroshi, Kazuto Suganuma, Masato Shikami, Norikazu Imai, Mayuko Sakai, Akihito Hiramatsu, Hidesuke Yamamoto, et al. "Energy Metabolism of Leukemia Cells: Glycolysis Vs Oxidative Phosphorylation." Blood 112, no. 11 (November 16, 2008): 2935. http://dx.doi.org/10.1182/blood.v112.11.2935.2935.
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Abstract Cancer cells are more dependent on glycolysis than oxidative phosphorylation in the mitochondria for generation of ATP as energy source. By using 2-deoxy-D-glucose (2-DG: glycolysis inhibitor) and oligomycin (inhibitor of oxidative phosphorylation), we examined the energy metabolism of various leukemia cell lines. The growth of the cell lines was measured by MTS assay, which detects viable cells in proliferation. 2-DG suppressed the growth of all leukemia cell lines examined in dose-dependent manners. The IC50 of each cell line was as follows: Kasumi-1 0.5±0.1mM, KG-1a 1.8±0.6mM, HL-60 3.3±0.1mM, NB4 3.8±0.4mM, and THP-1 23.1±3.8mM. The concentration of lactic acid (the final product of glycolytic pathway) in the culture supernatant was greatly reduced by the treatment with 0.2mM 2-DG for 24 hours in Kasumi-1 (54.5% of the control), compared with THP-1 (92.2%). It is suggested that the growth of Kasumi-1 was strongly suppressed by 2-DG through inhibition of glycolysis, which is supposed to be a main metabolic pathway in this cell line. On the other hand, treatment with oligomycin (1μg/ml) for 48 hours potently suppressed the growth of THP-1 (44.7%), then Kasumi-1 (72.1%). The growth of NB4, KG-1a and HL-60 was minimally suppressed (more than 90%) by oligomycin. Cell cycle was analyzed after 24 hours treatment with 2-DG or oligomycin. Sub-G1 fraction (apoptosis) was greatly increased by 2-DG (5mM) in Kasumi-1 (56.5%) and NB4 (30.6%), compared with THP-1 (7.6%). The apoptosis inducing effect was confirmed by annexinV staining. Oligomycin treatment (1μg/ml) increased apoptosis (subG1) in THP-1 (35.8%), then Kasumi-1 (16.6%) and NB4 (12.2%). Oligomycin treatment also increased G1 population (G1 arrest) in THP-1 (35.9% to 69.4%). AMP-activated protein kinase (AMPK) is activated by an elevated AMP/ATP ratio, which means the energy-deprived status of the cell. Western blot analysis using phospho-AMPK α (Thr172) antibody revealed that treatment with 2-DG or oligomycin induced prompt (30 min) phosphorylation of AMPK in leukemia cell lines. The extent of AMPK phosphorylation was almost proportional to the suppression of the growth. Collectively, it is suggested that leukemia cells are dependent almost exclusively on either glycolysis or oxidative phosphorylation in the mitochondria for energy production. Then, inhibition of glycolysis by 2-DG or oxidative phosphorylation by oligomycin results in growth suppression by inducing apoptosis and/or cell cycle arrest through activation of AMPK. Our data clarified the characteristics of the energy metabolism of each leukemia cell, and showed the key to produce novel therapeutic approach targeting metabolic pathway.
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Duque-Afonso, Jesus, Aitomi Essig, Leticia M. Solari, Tobias Berg, Heike L. Pahl, and Michael Lübbert. "Re-Expression of the AML1/ETO Target Gene LAT2/NTAL/LAB Results In Direct Interference with Myeloid Differentiation In AML1/ETO-Positive Cells." Blood 116, no. 21 (November 19, 2010): 2474. http://dx.doi.org/10.1182/blood.v116.21.2474.2474.
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Abstract Abstract 2474 Background: The leukemia-specific oncofusion protein AML1/ETO regulates different target genes, including the LAT2 gene (encoding the adaptor molecule LAT2/NTAL/LAB), which is epigenetically repressed by AML1/ETO as we have previously described. LAT2 is phosphorylated by c-kit and has a role in mast cell and B cell activation. To address the functional role of LAT2 during myeloid differentiation, expression studies were performed in myeloid cell lines, and LAT2 was overexpressed by retroviral gene transfer in AML1/ETO-positive Kasumi-1 cells and AML1/ETO-negative U-937 cells. Methods: To induce monocytic and granulocytic differentiation, the myeloid cell lines U-937, HL-60 and NB4 were treated with PMA and ATRA, respectively, and LAT2 expression measured by both Northern and Western blot. LAT2 was overexpressed in Kasumi-1 and U-937 cells by use of the retroviral vector pMYSiG-IRES-GFP. Virus was produced in 293T cells and titrated in TE671 cells. Following transduction, GFP-positive cells were sorted by fluorescence-activated cell sorting (FACS). Transduced cells were treated with PMA (2 and 10 nM for 24 and 48 hours) and ATRA (0.1 μM and 0.5 μM for 48 and 96 hours), respectively. Results: The AML1/ETO-negative myeloid cell lines HL-60, NB4 and U-937 readily expressed LAT2, which was further upregulated by PMA, and transiently downregulated with ATRA. In the AML1/ETO-positive Kasumi-1 and SKNO-1 cells, LAT2 expression was absent. To address the functional role of this repression, forced expression of LAT2 was achieved in Kasumi-1 and U-937 cells and resulted in effective processing of LAT2 protein (confirmed by Western blot), and a decrease in the expression of the differentiation markers CD11b and CD11c (FACS analysis) in Kasumi-1 but not U-937, with only minor effects of LAT2 overexpression upon apoptosis and cell growth arrest. Notably, during both PMA- and ATRA-induced differentiation, a striking maturation block occurred in Kasumi-1 (measured by CD11b/CD11c expression, observed at different doses and time points of these treatments), while differentiation of U-937 cells was unimpaired by overexpression of LAT2. Conclusions: In AML1/ETO-negative leukemia cells, LAT2 expression is differentially regulated during monocytic and granulocytic differentiation. In AML1/ETO-positive leukemia cells, in which LAT2 is repressed, LAT2 re-expression imposes a striking maturation block. Graded expression of this novel AML1/ETO target gene may therefore play an important role in maintaining the phenotypic characteristics of this leukemia subtype. Disclosures: No relevant conflicts of interest to declare.
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Gupta, Devansh, Somanath Tripathy, and Bodhisatwa Mazumdar. "Correlation Power Analysis of KASUMI and Power Resilience Analysis of Some Equivalence Classes of KASUMI S-Boxes." Journal of Hardware and Systems Security 4, no. 4 (October 16, 2020): 297–313. http://dx.doi.org/10.1007/s41635-020-00104-y.
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Adebayo, Ismail Abiola, Adamu Ibrahim Usman, Fatimah Bukola Shittu, Noor Zafirah Ismail, Hasni Arsad, Taoheed Kolawole Muftaudeen, and Mohammed Razip Samian. "Boswellia dalzielii-Mediated Silver Nanoparticles Inhibited Acute Myeloid Leukemia (AML) Kasumi-1 Cells by Inducing Cell Cycle Arrest." Bioinorganic Chemistry and Applications 2020 (September 22, 2020): 1–11. http://dx.doi.org/10.1155/2020/8898360.
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Background. Acute myeloid leukemia (AML) persists to be a major health problem especially among children as effective chemotherapy to combat the disease is yet to be available. Boswellia dalzielii is a well-known herb that is traditionally used for treatment and management of many diseases including degenerative diseases. In this study, silver nanoparticles were synthesized from the phytochemicals of B. dalzielii stem bark aqueous extract. The silver nanoparticles were characterized by carrying out Fourier Transform Infrared (FTIR) spectroscopy, Energy Filtered Scanning Electron Microscopy (FESEM), X-ray diffraction, and Dynamic Light Scattering (DLS) analyses. Antioxidant capacity of the nanoparticles was evaluated using 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, and the antiproliferative effect of the nanoparticles on Kasumi-1 leukemia cells was investigated using PrestoBlue assay. Flow cytometry analysis was performed to observe the effect of the nanoparticles on the leukemia cell cycle progression. Results. Our findings revealed that the synthesized silver nanoparticles were formed from electrons of the plant phytochemicals which include aromatic compounds, ethers, and alkynes. FESEM analysis revealed that the sizes of the nanoparticles range from 12 nm to 101 nm; however, DLS analysis estimated a larger average size of the nanoparticles (108.3 nm) because it measured the hydrodynamic radii of the nanoparticles. The zeta potential of the nanoparticles was −16 nm, and the XRD pattern of the nanoparticles has distinct peaks at 38.02°, 42.94°, 64.45°, 77.20°, and 81.47°, which is typical of face-centered cubic (fcc) structure of silver. The Trolox Equivalence Antioxidant Capacity (TEAC) of the nanoparticles was estimated to be 300.91 μM Trolox/mg silver nanoparticles. The nanoparticles inhibited Kasumi-1 cell proliferation. The half minimal inhibitory concentrations (IC50s) that inhibited Kasumi-1 cell proliferation are 49.5 μg/ml and 13.25 μg/ml at 48 and 72 hours, respectively. The nanoparticles induced cell cycle arrest in the Kasumi-1 cells at S (5% increase) and G2/M (3% increase) phases. Conclusion. The nanoparticles synthesized from the stem bark extract of B. dalzielii inhibit the growth of Kasumi-1 leukemia cells by activating cell cycle arrest; thus, they are potential antileukemic agents.
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Yu, Guopan, Ling Jiang, Changxin Yin, Zhixiang Wang, Hongsheng Zhou, Xuejie Jiang, Qifa Liu, and Fanyi Meng. "APP Gene Involves in the Regulation of Cell Apoptosis in AML1-ETO-Positive Leukemia Via SCF/c-Kit Signaling Pathway." Blood 126, no. 23 (December 3, 2015): 3647. http://dx.doi.org/10.1182/blood.v126.23.3647.3647.
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Abstract It is known that SCF/c-kit signaling pathway is one of the most important pathways in regulating of cell proliferation, differentiation and apoptosis, which can be continuously activated by C-KIT mutation and then lead to cell proliferation increase or apoptosis decrease. Furthermore, we have proven in our previous study that amyloid precursor protein (APP) gene, which is reported to increase cell proliferation in some solid cancer, is correlated with C-KIT mutation and adversely affects the disease outcome in AML1-ETO-positive acute myeloid leukemia (AML1-ETO-positive AML). In this study we focus on the regulation of cell proliferation and apoptosis in AML1-ETO-positive leukemia by APP gene and its mechanism. APP and C-KIT expression in bone marrow cells before the first chemotherapy from the 65 patients with AML1-ETO-positive AML (Blood. 2014,124:942) was simultaneously assessed by quantitative reverse transcriptase (QRT)-PCR method, meanwhile, the correlation of APP expression with peripheral WBC count, and the rate of bone marrow cellularity and blasts were observed. Furthermore, kasumi-1 cell line was chosen as cell model, and APP gene was knocked down using siRNA technology. The correlation of cell proliferation, differentiation and apoptosis with APP expression, as well as the regulation of SCF/c-kit signaling pathway by APP gene was analyzed on the cell line. WBC count and bone marrow cellularity, but not bone marrow blasts, was correlated with APP expression, in that a higher WBC count (29.2±3.9·109/L) was observed in APP-H patients when compared with 17.9±2.9·109/L in APP-L patients (P=0.008), and a higher percentage of bone marrow cellularity in APP-H patients (86.7%±1.7%) versus 80.3%±2.3% in APP-L cases (P=0.031). Moreover, the level of C-KIT mRNA expression was positively correlated with APP expression (r=0.349, P=0.011). In kasumi-1 cell line, compared with wild type and negative control cells, cell apoptosis, both early and late, increased significantly when APP gene was knocked down (Figure 1)., however, not apparently change was observed in cell proliferation and differentiation. What's more, C-KIT expression at both transcription (as evidenced by RTQ¨CPCR analysis) and translation (as confirmed by CD117 assay) levels, as well as BCL-2, C-KIT, p-ERK, p-AKT, P53 and NF-κB (as evidenced by western blot analysis, Figure 2), decreased significantly when APP was knocked down. In conclusion, APP gene involves in the regulation of cell apoptosis but not proliferation in AML1-ETO-positive leukemia via SCF/c-kit signaling pathway. Figure 1. Flow cytometry analysis of cell apoptosis in kasumi-1 cells with different APP expression. Both the rate of early (Q4-2) and late apoptosis (Q2-2) in si-APP kasumi-1 cell increased significantly when compared with the rate in wild type and NC kasumi-1 cell (Early apoptosis rate: si-APP: 29.00%±0.98%, wild type: 21.43%±0.86%, NC: 21.67%±0.78%, F=71.927, P<0.001; Late apoptosis rate: si-APP: 19.80%±1.51%, wild type: 12.33%±0.75%, NC: 12.90%±1.25%, F=35.239, P<0.001). Figure 1. Flow cytometry analysis of cell apoptosis in kasumi-1 cells with different APP expression. Both the rate of early (Q4-2) and late apoptosis (Q2-2) in si-APP kasumi-1 cell increased significantly when compared with the rate in wild type and NC kasumi-1 cell (Early apoptosis rate: si-APP: 29.00%±0.98%, wild type: 21.43%±0.86%, NC: 21.67%±0.78%, F=71.927, P<0.001; Late apoptosis rate: si-APP: 19.80%±1.51%, wild type: 12.33%±0.75%, NC: 12.90%±1.25%, F=35.239, P<0.001). Figure 2. Western blot analysis of the expression of BCL-2, C-KIT, p-ERK, p-AKT, P53 and NF-κB with different APP expression. Figure 2. Western blot analysis of the expression of BCL-2, C-KIT, p-ERK, p-AKT, P53 and NF-κB with different APP expression. Disclosures No relevant conflicts of interest to declare.
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Fuchs, Ota, Dana Provaznikova, Gabriela Peslova, Marcela Kocova, Iuri Marinov, and Ivan Spicka. "Differential Induction of Apoptosis in Human Leukemia Cells (ML-1, ML-2, CTV-1 and KASUMI-1) Exposed to the Proteasome Inhibitor Bortezomib and the Effect of Transforming Growth Factor-beta Signaling Pathway." Blood 108, no. 11 (November 16, 2006): 4359. http://dx.doi.org/10.1182/blood.v108.11.4359.4359.
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Abstract Bortezomib (N-pyrazinecarbonyl-L-phenylalanine-L-leucine boronic acid; VELCADE, formely known as PS-341 or MLN-341; Millenium Pharmaceuticals, Inc., Cambridge, MA) is a potent and specific proteasome inhibitor. It is the first member of proteasome inhibitors which obtained approval as chemotherapeutic agent for the treatment of relapsed or refractory multiple myeloma. First clinical experience of bortezomib in patiens with follicular lymphoma and mantle cell non-Hodgkin lymphoma suggest that bortezomib is well tolerated and has significant single-agent activity. Several phase I trials on the use of bortezomib as a novel treatment strategy in leukemia have been started. We studied the effect of of bortezomib and of transforming growth factor-beta (TGF-β) on induction of cell cycle arrest and apoptosis in human leukemia cells (ML-1, ML-2, CTV-1 and KASUMI-1) established from the peripheral blood of patients with acute myeloid leukemia (AML). [6-3H] thymidine incorporation was used as a measure of DNA synthesis to estimate cell proliferation. Apoptosis was detected by flow cytometry using annexin V-FITC/propidium iodide assay and results were confirmed by cell cycle analysis. The profiles of cellular DNA contents indicated the distribution of the cells in different phases of the cell cycle and any possible DNA loss due to DNA fragmentation during apoptosis. The cells with a DNA content less than that of G1 cells were considered as apoptotic cells. Statistical significance of the experimental results was analyzed by Student’s paired t-test. Leukemia cells were preincubated for 24–96 h without addition (control) or with bortezomib (4 nM or 10 nM) or with TGF-β1 (5 ng/ml or 10 ng/ml). TGF-β1 inhibited DNA synthesis only in KASUMI-1 cells but not in other leukemia cells used. Bortezomib (10 nM) was potent inhibitor of DNA synthesis in all four types of leukemia cells and induced apoptosis in KASUMI-1, ML-2 and CTV-1 cells but not in ML-1 cells [Figure 1]. Kinetics of apoptosis was different in individual cell lines. The peak of apoptosis was reached in 24 h in ML-2 cells, however in KASUMI-1 and CTV-1 cells in 48h. KASUMI-1 cells were most sensitive to bortezomib. In addition KASUMI-1 and ML-2 cells were also sensitive to induction of apoptosis by TGF-β1 but in lesser extent than by bortezomib [Figure 1]. Kinetics of apoptosis induction by TGF-β1 was slower than with bortezomib and lasted 48–96 h. Different sensitivity of human leukemic cell lines to bortezomib likely mimics behaviour of primary leukemic cells of patients and thus limits the use of proteasome inhibitors in therapy. Figure 1 Figure 1.
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Miwa, Hiroshi, Masato Shikami, Norikazu Imai, Kazuto Suganuma, Mineaki Goto, Shohei Mizuno, Miyuki Takahashi, and Masakazu Nitta. "Some Leukemia Cells Are Dependent on Glutamine as Energy Source." Blood 116, no. 21 (November 19, 2010): 4861. http://dx.doi.org/10.1182/blood.v116.21.4861.4861.
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Abstract Abstract 4861 In cancer cells, glucose uptake is elevated and glycolysis persists even under aerobic conditions (Warburg effect). Glutamine metabolism is another target for alteration in cancer development. Glutaminolysis (catabolism of glutamine to generate ATP) is known to increase in tumors. We examined the dependency of the leukemia cells (Kasumi-1, THP-1, HL-60 and NB4) on glucose or glutamine by measuring the growth (MTS count) in glucose- or glutamine-deprived condition. Glucose withdrawal greatly suppressed the growth of all 4 cell lines. However, glutamine withdrawal showed different growth suppressive effects among the cell lines (Kasumi-1: 55% of control, THP-1: 60%, HL-60: 39%, NB4: 70%). HL-60 was most sensitive to glutamine deprivation. The growth suppression of HL-60 due to glutamine withdrawal was partially rescued by oxaloacetate (OAA), a TCA cycle metabolite, while the growth of other cell lines was not rescued by OAA. In the course of glutamine catabolism, ammonia is liberated. Although basal level of the ammonia concentration was not so different among each cell line, glycolysis inhibitor (2-deoxyglucose) treatment enhanced the ammonia generation in HL-60 (Kasumi-1: 2.8% increased, THP-1: 1.7%, HL-60: 6.1%, NB4: 2.8%). Glutaminase, an enzyme converting glutamine to glutamate, is most abundantly expressed in HL-60 in western blot analysis. In addition, HL-60 was most sensitive to the treatment with aminooxyacetate, an inhibitor of glutamate-dependent transaminases that convert glutamate into a-ketoglutarate in the glutaminolytic pathway (Kasumi-1: 86% of control, THP-1: 97%, HL-60: 79%, NB4: 83%). Taken together, HL-60 was considered as glutamine dependent cell line. Therapies targeting glutamine metabolism, such as glutamine depletion or use of inhibitor of glutaminolytic pathway, might be effective against some leukemia. Disclosures: No relevant conflicts of interest to declare.
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Heidenreich, Olaf, Jürgen Krauter, Heidemarie Riehle, Philipp Hadwiger, Matthias John, Gerhard Heil, Hans-Peter Vornlocher, and Alfred Nordheim. "AML1/MTG8 oncogene suppression by small interfering RNAs supports myeloid differentiation of t(8;21)-positive leukemic cells." Blood 101, no. 8 (April 15, 2003): 3157–63. http://dx.doi.org/10.1182/blood-2002-05-1589.
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Abstract The translocation t(8;21) yields the leukemic fusion gene AML1/MTG8 and is associated with 10%-15% of all de novo cases of acute myeloid leukemia. We demonstrate the efficient and specific suppression of AML1/MTG8 by small interfering RNAs (siRNAs) in the human leukemic cell lines Kasumi-1 and SKNO-1. siRNAs targeted against the fusion site of the AML1/MTG8 mRNA reduce the levels of AML1/MTG8 without affecting the amount of wild-type AML1. These data argue against a transitive RNA interference mechanism potentially induced by siRNAs in such leukemic cells. Depletion of AML1/MTG8 correlates with an increased susceptibility of both Kasumi-1 and SKNO-1 cells to tumor growth factor β1 (TGFβ1)/vitamin D3–induced differentiation, leading to increased expression of CD11b, macrophage colony-stimulating factor (M-CSF) receptor, and C/EBPα (CAAT/enhancer binding protein). Moreover, siRNA-mediated AML1/MTG8 suppression results in changes in cell shape and, in combination with TGFβ1/vitamin D3, severely reduces clonogenicity of Kasumi-1 cells. These results suggest an important role for AML1/MTG8 in preventing differentiation, thereby propagating leukemic blast cells. Therefore, siRNAs are promising tools for a functional analysis of AML1/MTG8 and may be used in a molecularly defined therapeutic approach for t(8;21)-positive leukemia.
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Meng, Fan Yi, Ling Jiang, Qingxiu Zhong, Li Chun Wang, Guopan Yu, and Xinhua Zhu. "Molecular Mechanisms of EML in AML1/ETO+ AML and Its Targeting Treatments." Blood 124, no. 21 (December 6, 2014): 5953. http://dx.doi.org/10.1182/blood.v124.21.5953.5953.
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Abstract Our previous study has been reported that AML1/ETO positive patients with highly expressed of APP were much easier to extramedullary infiltration, and got poor prognosis£¨followed-up median 35 ( 6-96 ) months, RFS in the high expression APP group was significantly lower than the low expression group £¬40.0% vs 80.0%£¬P = 0.001). In vitro study, we constructed a cell model kasumi-1 which was consistent with AML1/ETO positive and high expressed of APP gene. The cell migration was significantly reduced after interferce of APP expression. This study was designed to investigate the molecule mechanism of extramedullary leukemia (EML) in kasumi-1 cell model and to invent a strategy for treatment in clinic. In this study, we found p-ERK, c-Myc and MMP-2 were significantly decreased after APP expression knockdown in kasumi-1 cell. Meanwhile, we added the inhibitors to block p-ERK and c-Myc expression. The results showed that protein expression of p-ERK and c-Myc was significantly decreased after p-ERK inhibitor performance, which was proportional to the time and concentration. c-Myc and MMP-2 protein expression was significantly reduced after c-Myc inhibitor was used, but p-ERK didn't change (Fig.1B). So, we concluded that APP might regulated the AML cell migration via APP/p-ERK/c-Myc/MMP-2 pathway. Also, we found that kasumi-1 cell was resistant to adriamycin (ADM) and Ara-C, which meant APP may be related with drug resistance. So, we detected cell surviving fraction after ADM and Ara-C performance via MTT assay. The results showed that there was no difference in control group and siAPP group. But, when compared with controls groups, cell surviving fraction in siEZH2 group was significantly decreased after ADM and Ara-C performance respectively. Furthermore, we found protein expression of EZH2 was significantly reduced after LBH589 treatment in cell culture. So, we concluded that LBH589 or SiEZH2 could increase sensitivity of kasumi-1 cell to ADM and Ara-C. In sum, in AML1/ETO positive leukemia cells, we first report that APP gene regulates cell migration via APP/p-ERK/c-Myc/MMP-2 pathway and EZH2 gene induces drug resistantence. Interference or blocking of EZH2 and APP expression may be helpful in treating AML1/ETO positive leukemia. Disclosures No relevant conflicts of interest to declare.
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Gasparetto, Maura, Craig T. Jordan, Mohammad Minhajuddin, Daniel A. Pollyea, Vasilis Vasilou, Philip Reigan, R. Keith Humphries, and Clayton A. Smith. "ALDH Genes and Reactive Aldehydes Play Important Roles in HSCs and Leukemia and May Be Exploited to Treat AML." Blood 122, no. 21 (November 15, 2013): 2893. http://dx.doi.org/10.1182/blood.v122.21.2893.2893.
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Abstract ALDH1A1 is expressed at high levels in normal HSCs and we previously reported that its function might involve metabolism of compounds termed reactive aldehydes. We also reported that loss of ALDH1A1 led to a compensatory increase in a related isoform, ALDH3A1, which also metabolizes reactive aldehydes. Double knockouts for both ALDH1A1/3A1 accumulate reactive aldehydes, which appear to impact a number of cellular processes including signal transduction and gene expression. As reactive aldehydes also cause DNA damage, we hypothesized that excess accumulation of reactive aldehydes may predispose to leukemic transformation of HSCs. In support of this, we found that ALDH1A1/3A1 double knockout HSCs readily form acute leukemia following transduction with a NUP98-HOXA10 fusion gene, which rarely causes leukemic transformation in wild type HSCs. Furthermore, in human AML, frequent absence of ALDH1A1 and the universal absence of ALDH3A1 was observed. A human AML cell line, Kasumi-1, was found to be ALDH1A1/3A1 deficient and to have high levels of intracellular reactive aldehydes. In addition, Kasumi-1 was highly sensitive to DNA damage and cell death following exposure to exogenous 4-HNE, a prototypic reactive aldehyde. In contrast, normal CD34+ HSCs were relatively resistant to 4-HNE. Based on these observations, we further hypothesized that treatment of ALDH1A1/3A1 deficient AMLs with clinically relevant compounds that further increase intracellular 4-HNE levels would selectively eliminate AML while sparing normal CD34+ HSCs. To test this, Kasumi-1 were exposed to a series of compounds including the pro-oxidant Arsenic tri-oxide (ATO), the sesquiterpene lactone parthenolide (PTL) and 4-HC, the active metabolite of cyclophosphamide (Cy) and a substrate of ALDH1A1. All increased intracellular 4-HNE levels and DNA damage. Exposure to combinations of 4-HC, ATO and PTL induced high levels of cell death in Kasumi-1. In contrast, Kasumi-1 cells engineered to express ALDH1A1 through lentiviral gene transfer and normal CD34+ HSCs were relatively resistant to several of these treatments. Primary ALDH1A1/3A1- AMLs were also relatively sensitive to treatment with these same compounds. In conclusion, ALDHs and reactive aldehydes may play important roles in HSCs and leukemia and exploitation of their biology may lead to novel therapies for AML and possibly other cancers. As an initial application of this treatment strategy, we are developing a clinical trial to treat patients with relapsed/refractory ALDH1A1/3A1 deficient AML with Cy/ATO. Disclosures: No relevant conflicts of interest to declare.
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Suh, Hyung Chan, Katherine Pohl, Anna Patricia L. Javier, Dennis J. Slamon, and John P. Chute. "Effect of dendritic cells (DC) transduced with chimeric antigen receptor (CAR) on CAR T-cell cytotoxicity." Journal of Clinical Oncology 35, no. 7_suppl (March 1, 2017): 144. http://dx.doi.org/10.1200/jco.2017.35.7_suppl.144.
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144 Background: T cells interacting DC could be superior in T cell cytotoxicity. CD141+/Cleg9a+ intra-tumoral DC play a critical role in tumor cytotoxicity. Therefore, combining intra-tumoral DC in CAR T cell would safely increase localized CAR T cell cytotoxicity. We hypothesized that bioengineered DC compartment could be an excellent source for enhanced CAR T cell cytotoxicity. Methods: DC precursors and T cells of PBMC were transduced with a CAR (pCCL-anti-CD33-4-1BB-CD3z-T2A-GFP; CAR-DC or CAR T). For comparison, additional DC were transduced with 4-1BB cDNA (pCCL-4-1BB-T2A-GFP; 4-1BB-DC) or mock control (pCCL-eGFP). In addition to lentivirus transduction, differentiation of DC in vitro employed Flt3L/GM-CSF/IL-4. Transduced CAR T and CAR-DC were sorted by GFP expression at day 5. After further 10 days of culture, cells were harvested and analyzed for phenotype. An acute myeloid leukemia (AML) cell line (Kasumi-1) was treated with CAR T +/- CAR-DC, 4-1BB-DC, or mock control for functional assays. Results: Frequencies of cells expressing CD141+/Cleg9a+ were higher in 4-1BB-DC vs. control DC (33% vs. 1.5%). After mixing CAR T and CAR-DC (5X105) with Kasumi-1 (1X105) for 6 hours, CAR T/CAR-DC showed 100% Kasumi-1 cell cytotoxicity compared to 70% of CAR T by trypan blue. CAR T/CAR-DC also demonstrated higher Annexin V positive Kasumi-1 cells compared with CAR-T (91% vs. 52%). CAR T with or without CAR-DC were also assessed with multiplex immunoassays. CAR T cells mixed with CAR-DC induced higher level of IFN-gamma (10,316 vs. 6,186 pg/ml), IL-2 (68,840 vs. 64,708 pg/ml), and TNFalpha (1,361 vs. 905 pg/ml) (Kasumi-1 cells mixed with CAR-T cells of 10 E/T ratio) than CAR T cells. CAR-DC produced significantly higher IL-12 cytokine production (1,352 vs. 161 pg/ml) than CAR T cells in response to CD33 but independent to T cells, confirmed by comparing IL-12 production with CAR T/4-1BB-DC. Conclusions: These data show that in vitro differentiation of DC bearing 4-1BB increases CD141+/Cleg9a+ DC population and that interaction with CAR-DC to CAR T cells enhances anti-AML cytotoxicity. Our finding may implicate the development of CAR-DC therapy combined to CAR T cells to increase the efficiency of cancer immunotherapy.
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Kobune, Masayoshi, Kazuyuki Murase, Satoshi Iyama, Tsutomu Sato, Rishu Takimoto, Yutaka Kawano, Shohei Kikuchi, Koji Miyanishi, Yasushi Sato, and Junji Kato. "Cyclopamine Induced Apoptosis in Primary CD34+ Acute Leukemic Cells." Blood 114, no. 22 (November 20, 2009): 3768. http://dx.doi.org/10.1182/blood.v114.22.3768.3768.
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Abstract Abstract 3768 Poster Board III-704 Aberrant reactivation of Hedgehog (Hh) signaling has been described in a wide variety of human cancers and its role in maintenance of self-renewal of cancer stem cells. However, it is unclear whether Hh signaling contributes to the growth, survival and drug resistance of acute myeloid leukemic (AML) cells. Regarding normal CD34+hematopoietic cells, we previously showed that Indian Hh (Ihh) and its receptor molecules, Patched and Smoothened are expressed in cord blood (CB) CD34+ cells and Ihh regulate proliferation of short-term hematopoietic repopulating cells in vivo. In the present study, we assessed the possibility that Hh pathway activation contributes to survival and drug resistance of acute myeloid leukemic (AML) cells. Hh signaling in bone marrow (BM) CD34+ cells, leukemic cell lines and primary CD34+ leukemic cells were screened by RT-PCR and a Hh signaling reporter assay. We have found that Ihh were expressed in normal BM CD34+ cells, primary CD34+leukemic cells and most of human AML cell lines, HL60, U937, KG1, Kasumi-1, Kasumi-3 and TF-1. In contrast, Hh receptor components, Patched and Smoothened, were detected in BM CD34+ cells, primary CD34+leukemic cells (n=3) and cytokine responsible CD34+ AML cell lines such as Kasumi-1, Kasumi-3 and TF-1. Moreover, the downstream transcription factor GLI1 or GLI2 were expressed in BM CD34+ cells, primary CD34+leukemic cells and these CD34+ cell lines, indicating that Hh signaling was active in these cells. We further assessed whether Hh signaling transmit to GLI1 using GLI1-responsive luciferase assay. GLI-responsive reporter plasmid (TK-6GBS-Luc) was transduced into these cells in the presence or absence of anti-Hh neutralizing antibody 5E1. TK-6GBS-Luc-transduced Kasumi-1, Kasumi-3 and TF-1 cells showed high luciferase activity. Furthermore, the luciferase activity of these cells was significantly reduced in the presence of 10 μg/ml antibody 5E1. These results clearly indicated that Hh signaling could transmit into these CD34+ leukemic cell lines in autocrine manner. We next examined the effect of Hh inhibitors on these CD34+ leukemic cells. Inhibition of Hh signaling with the naturally derived chemical Smoothened antagonist Cyclopamine, endogenous Hh inhibitor Hedgehog-interacting protein or anti-Hedgehog neutralizing antibody induces apoptosis in these cells after 48 hr exposure although these CD34+ cell lines exhibit resistance to cytarabine (Ara-C) exposure. In contrast, cyclopamine did not affect growth and survival of the U937 or HL-60 cell line that lacks expression of Hh receptor components. Furthermore, combination of 10 μM cyclopamine significantly reduced the drug resistance against Ara-C in CD34+ cell lines as well as primary CD34+AML cells (Figure). These results suggest that Hh pathway activation is a feature of CD34+ myeloid leukemic cells and inhibition of Hh signaling pathway in combination with Ara-C etc. may be a new therapeutic strategy to eradicate CD34+ AML stem cells. Disclosures: No relevant conflicts of interest to declare.
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Forster, Victoria J., Patricia Garrido Castro, Amy K. Bradburn, James M. Allan, and Olaf Heidenreich. "The Angiogenic Factor Angiopoietin-1 Is Regulated by the Acute Myeloid Leukemia Fusion Protein AML1/ETO." Blood 118, no. 21 (November 18, 2011): 2426. http://dx.doi.org/10.1182/blood.v118.21.2426.2426.
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Abstract Abstract 2426 BACKGROUND The chromosomal rearrangement t(8;21)(q22;q22) encodes the fusion gene AML1/ETO, which is the most common translocation in acute myeloid leukemia (AML). It has an incidence of approximately 15% and a favourable prognosis in comparison to other AML subtypes. Dysregulated angiogenesis in the bone marrow niche environment is predicted to have an important role in leukemia pathogenesis, and several factors have been implicated in this process. Angiopoietin-1 (ANGPT1) is a cytokine involved in hematopoietic stem cell quiescence and regulation of microvessel density within the bone marrow, as well as having a role in transendothelial migration. Moreover, ANGPT1 is upregulated in leukemic blast cells from AML patients. In this study we investigated the role of AML1/ETO as a regulator of ANGPT1 expression, as well as functional implications of ANGPT1 in AML1/ETO-positive AML. METHODS In order to investigate putative AML1/ETO-dependent regulation of ANGPT1, we performed gain-of-function studies using lentiviral gene transfer to ectopically express AML1/ETO in HL-60 and U937 AML cell lines. We also depleted AML1/ETO in the t(8;21)-positive AML cell line Kasumi-1 using fusion gene specific siRNA. Additionally, the functional role of ANGPT1 was studied using targeted RNAi in Kasumi-1 cells. Transcript expression of AML1/ETO and ANGPT1 was analysed by quantitative Real-Time PCR (qRT-PCR) and AML1/ETO protein expression was quantified by western blotting. Angiopoietin-1 protein secretion was determined using enzyme-linked immunosorbent assay (ELISA). We also utilised Matrigel transwell assays to test the effect of ANGPT1 downregulation on the invasive and migratory properties of Kasumi-1. RESULTS In HL-60 and U937 transduced with AML1/ETO, we observed an up to 280 fold increase in ANGPT1 mRNA transcript levels as measured by qRT-PCR, which correlated with an increase in secreted Angiopoietin-1 protein. Conversely, siRNA-mediated AML1/ETO depletion in Kasumi-1 cells significantly decreased ANGPT1 transcript and protein levels after a single electroporation. After three serial electroporations with siRNA, AML1/ETO transcript levels were reduced by 85%, with a concomitant decline in ANGPT1 transcript (>99%) and secreted protein. Preliminary data suggest siRNA targeting of ANGPT1 in Kasumi-1 decreases the invasive ability of these cells, causing a ≥50% reduction in invasion when compared to controls. CONCLUSIONS We have identified a strong correlation between AML1/ETO and ANGPT1 expression, whereby a reduction of AML1/ETO results in a substantial reduction of ANGPT1. Similarly, the introduction of AML1/ETO into myeloid cell lines results in a large upregulation of ANGPT1. Preliminary evidence suggests that a reduction of ANGPT1 reduces the invasive and migratory potential of Kasumi-1. This could have major functional consequences in the bone marrow niche with regards to understanding the AML stem cell and its interaction with the niche environment as well as providing insight into how leukemic cells in the circulation might interact with the vasculature. Disclosures: No relevant conflicts of interest to declare.
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Nagamachi, Akiko, Hiroya Asou, Hirotaka Matsui, Yuko Ozaki, Daisuke Aki, Kazuko Miyazaki, Norimasa Yamasaki, et al. "Haploinsufficiency and Deficiency of a 7q Gene Titan (Samd9L) Predispose Leukemia Development in Cooperation with Deregulated Expression of a Transcription Factor, Evi1, or a Histone Demethylase, Fbxl10." Blood 112, no. 11 (November 16, 2008): 197. http://dx.doi.org/10.1182/blood.v112.11.197.197.
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Abstract In attempts to isolate myeloid tumor-suppressor genes responsible for 7q deletion, we identified a common microdeletion cluster in chromosome subband 7q21.2 by microarraybased CGH analyses of JMML (ASH Annual Meeting, 2006). This region was also deleted in nearly 30% of unselected adult MDS/AML patients, mostly as a part of monosomy 7 or larger 7q deletions. In this region, there are three poorly-characterized genes (Miki = LOC253012, Kasumi = Samd9, and Titan = Samd9L). Miki encoding a centrosomal protein is likely involved in myelodysplasia and chromosomal instability, which are characteristic of -7/7q- MDS/AML, as is presented in this meeting elsewhere. Kasumi (Samd9) and Titan (Samd9L) are related genes that encode 60% homologous proteins. Neither Kasumi nor Titan has homology with any other proteins or contain known functional motifs. Kasumi and Titan were ubiquitously expressed at a relatively constant level. However, in six cell lines derived from MDS/AML patients harboring monosomy 7, Kasumi protein was barely detectable, whereas Titan expression levels were roughly half of those in other AML cells. The mouse genome contains only Titan and lacks Kasumi gene, suggesting that the function of these two gene products are overlapping. We started to characterize these genes by generating mice deficient in Titan (titan−/−). titan−/− mice appear normal and no hematological abnormalities have been observed, suggesting that additional gene alterations are required for leukemia development. To address this issue, retroviral insertional mutagenesis was applied to the mice. Virus infection induced acute leukemia in homozygous (titan−/−) and heterozygous (titan+/−) mice with higher morbidity and mortality than in wild-type (titan+/+) littermates. Leukemias developed in titan+/+ mice were mainly of T-cell lineage. By contrast, those developed in titan−/− and titan+/− mice were negative for lymphoid markers but expressed various combination of cell surface markers for myeloid (Gr1), monocytic (Mac1), erythyroid (Ter119) and megakaryocytic (CD61) progenitors. Histopathology demonstrated that leukemia cells infiltrated the liver, lung, kidneys and spleen, and a portion of the infiltrated cells were maturated. These data suggests that leukemias that developed in titan-deficient mice represent stem cell malignancy rather than AML. Inverse PCR detected two common integration sites (CIS) specific for titan−/− and titan+/− mice, which induced deregulated expression of a zinc finger transcription factor, Evi1, and a histone demethylase, Fbxl10. In addition, although it was not a CIS, TGFβ was isolated as a major viral integration site in one tumor. These results demonstrated that haploinsufficiency and deficiency of Titan predispose leukemia development through inhibition of TGFβ-mediated signaling or an epigenetic change. Recently, deleterious mutations in the Titan gene were reported to be involved in Normophosphatemic Familial Tumoral Carcinosis, a rare autosomal recessive disease in five families of Jewish-Yemenite origin. Impairment of cell migration is suspected to be a cause of this disease and, indeed, wound healing test revealed that fibroblasts established from titan−/− and titan+/− mice migrate slower than those established from wild-type mice. Relevance of the impairment of cell migration to development of leukemia in titan-deficient mice is currently under investigation.
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van Luijn, Marvin M., Martine E. D. Chamuleau, Theresia M. Westers, James A. Thompson, Suzanne Ostrand-Rosenberg, Gert J. Ossenkoppele, S. Marieke van Ham, and Arjan A. Van de Loosdrecht. "Class-II Associated Invariant Chain Peptide (CLIP) Expression on AML Blasts Adversely Affects Alloreactive CD4+ T Cell Recognition." Blood 110, no. 11 (November 16, 2007): 4888. http://dx.doi.org/10.1182/blood.v110.11.4888.4888.
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Abstract Although acute myeloid leukemia (AML) can be cured with intensive treatment including myeloablative chemotherapy and haematopoietic stem cell transplantation, relapses occur in the majority of cases. A common feature of tumor cells is their ability to escape immune surveillance through adapted intrinsic mechanisms. Thus, it is a great challenge to develop optimal strategies that direct a specific cellular immune response against residual AML blasts in vivo. As CD4+ T cells are needed to initiate a strong anti-leukemic CD8+ T cell response, the mechanism through which HLA class-II restricted (leukemia-specific) antigens are presented on AML blasts could be an essential factor in immune surveillance. Previously, we showed that the self peptide Class-II Associated Invariant Chain Peptide (CLIP) important in HLA class-II antigen presentation appeared to be disadvantageous, as its expression on AML blasts predicted a shortened disease-free survival (Chamuleau et al. Canc. Res.2004; 64(16):5546–50). We hypothesized that CLIP interferes with the presentation of specific tumor antigens on HLA class-II molecules, thereby preventing recognition of AML blasts by CD4+ T cells. To investigate whether CLIP expression indeed has a functional effect on leukemia-specific T cell activation in patients, an AML cell line model with CLIP+ and CLIP− leukemic blasts was set up. The Kasumi-1 and THP-1 AML cell lines were selected as both stained positive for extracellular HLA-DR (89%; MFI=31.3 and 91%; MFI=37.5 respectively) and CLIP expression (88%; MFI=37.2 and 91%; MFI=34.0 respectively) by flow cytometric analysis. These DR+CLIP+ cell lines were specifically silenced for Invariant Chain (Ii) expression using RNA interference to down-modulate CLIP presentation on the cell surface. Indeed, Ii siRNA-treated cells not only showed a significant decrease of intracellular Ii expression (MFI decrease of 87.7% for Kasumi-1 and 82.7% for THP-1), but also a marked downregulation of relative CLIP amount per HLA-DR molecule (fold decline in CLIP/DR ratio of 1.4 for Kasumi-1 and 2.0 for THP-1). Wild type (DR+CLIP+) and modulated (DR+CLIP−) cells of Kasumi-1 or THP-1 origin acted as stimulators for alloreactive CD4+ T cells in mixed leukocyte reactions using different stimulator to responder (S/R) ratios. Modulated DR+CLIP− Kasumi-1 and THP-1 cells induced a strong increase in alloreactive CD4+ T cell proliferation as compared to DR+CLIP+ wild type controls, both in an HLA-DR-specific and a S/R-dependent manner. At the highest S/R ratio, mean proliferation increases of 2.58-fold for Kasumi-1 (n=3) and 1.71-fold for THP-1 (n=2) were observed. These data support our hypothesis that the expression of CLIP on AML blasts plays an important role in immune surveillance, which might have impact on cellular immunotherapy with dendritic cell-based vaccines in AML.
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Tabe, Yoko, Rooha Contractor, Susanne Radke, Michael Andreeff, and Marina Konopleva. "Novel Role of HDAC Inhibitors in Bone Marrow Microenvironment: Activation of Leukemia Cell Phagocytosis through Annexin A1." Blood 106, no. 11 (November 16, 2005): 2219. http://dx.doi.org/10.1182/blood.v106.11.2219.2219.
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Abstract Annexin A1 (ANX-A1) is a calcium-dependent membrane-binding protein involved in the modulation of apoptosis and phagocytosis (FASEB J.2003;17:1544). We have previously reported that HDAC inhibitor depsipeptide (FK228) caused marked growth inhibition and apoptosis in t(8;21) Kasumi-1 AML cells with up-regulation of 123 genes (by cDNA array) including ANX-A1 (3.5 fold; Tabe, Blood 2004). By chromatin immunoprecipitation (ChIP) assay, FK228 induced H4 and H3-K9 acetylation in the ANX-A1 promoter with corresponding induction of ANX-A1 mRNA (7.2±1.7 fold, TaqMan RT-PCR) and protein (western blot analysis). The markedly increased ANX-A1 protein localized on the cell membrane of Kasumi-1 cells exposed to FK228 was confirmed by immunofluorecence analysis using confocal microscopy. ANX-A1 membrane localization was diminished by treatment with anti-ANX-A1 mAb. To investigate the contribution of ANX-A1 to FK228-induced apoptosis, we neutralized ANX-A1 by anti-ANX-A1 mAb. This moderately decreased FK228 induced apoptosis (36.0±4.1 vs 26.5±3.7% AnnexinV(+)/PI(+) cells, p=0.01). Similarly, Kasumi-1 cells transfected with siRNA/ANX-A1 were less sensitive to FK228-induced cell death compared with nonsense (N) siRNA transfected cells (siRNA 31.2±3.1% vs NsiRNA 39.5±2.9% annexin(+) cells, p=0.03). These data indicate that the upregulation of endogeneous ANX-A1 (either membrane-binding or secreted form) promotes cell apoptosis in an autocrine fashion. Next, we investigated the functional role of ANX-A1 on leukemia cell phagocytosis. The engulfment of Kasumi-1 cells by cocultured human THP-1 monocyte-derived macrophages was evaluated by cell adherence assay. Compared with untreated cells, the exposure to FK228 induced a dramatic increase in Kasumi-1 cells attachment to macrophages (untreated vs FK228 treated; 57 ± 9 cells vs 196 ± 33 cells/ microscopic fields (0.08 mm2/field), n = 5; p=0.01). FK228-induced cell attachment was completely abrogated in the siRNA/ANX-A1 transfected Kasumi-1 cells (60.5% ± 10.5% decrease; n = 5; p<0.001). Consistently, co-treatment with FK228 and anti-ANX-A1 mAb followed by washout of both compounds resulted in significantl repression of FK228-stimulated engulfment of leukemic cells by macrophages (54.1% ± 3.0% decrease; n = 5; p=0.02). This effect was not further enhanced by adding anti-ANX-A1 mAb to the co-culture medium, suggesting that membrane-associated but not soluble ANX-A1 contributes to leukemia cell engulfment by macrophages. Results presented here demonstrate a novel mechanism of action of HDAC inhibitors in the context of bone marrow microenvironment via histone acetylation, increased expression and externalization of ANX-A1, which provides an “eat-me” signal and mediates phagocytic clearance of apoptotic leukemic cells by macrophages. Our data further suggest that ANX-A1 is silenced via histone deacetylation in leukemic cells, and its re-expression by HDAC inhibitors may stimulate apoptosis in an autocrine fashion while diminishing the inflammatory response through activating phagocytosis in the bone marrow microenvironment.
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SENOO, Hiroshi, and Tadaharu ISHIKAWA. "Hydraulic function of the kasumi levee system on the Kurobe Alluvial Fan of the 19th century." E3S Web of Conferences 40 (2018): 06032. http://dx.doi.org/10.1051/e3sconf/20184006032.
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Kasumi levees are a type of discontinuous levee system that was used in early-modern times of Japan, but few records remain of the hydraulic design of the levee construction. Results of numerical flow simulation are presented for the hydraulic functioning of the Kasumi levee system along the Kurobe River that flows on a steep alluvial fan. Historical records from the 19th century to the present were used to simulate the flow through a levee system.. The computational results suggest that the flood control strategy was such that old river paths were utilized for temporary floodways and a portion of this diverted flow was returned to the main river channel through funnel-shaped levee openings located along the middle reach to prevent inundation of the alluvial fan.
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Wu, Kangni, Yongxian Hu, Huarui Fu, Lixia Sheng, and He Huang. "Ex Vivo-Expanded Vγ9Vδ2 T Cells Can Efficiently Kill Human Acute Myeloid Leukemia Cells Via Trogocytosis." Blood 118, no. 21 (November 18, 2011): 580. http://dx.doi.org/10.1182/blood.v118.21.580.580.
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Abstract Abstract 580 Cellular immunotherapy of hematopoietic malignancies is regarded as one of the most promising approaches to deal with the common relapse or resistance to conventional treatments. γδT cells are innate-like lymphocytes capable of potent antitumor activity toward a variety of malignant cell types and have been applied to clinical trials with positive effects. But it still lacks basis whether these cells coule be used in the treatment of acute myeloid leukemia(AML).Here we set out to determine the potent cytotoxicity of ex vivo-expanded Vγ9Vδ2 T cells against AML cells and the underlying mechanisms. To expand human Vγ9Vδ2 T cells, peripheral blood mononuclear cells(PBMCs)from healthy donors(HD)were cultured in the presence of 2uM zoledronate together with recombinant human IL-2 ( 250 IU/ml). After culture in vitro for 12 days, 90–95% Vγ9Vδ2 T cells were detected by flow cytometry(FACS). Cell viability was determined using trypan blue exclusion and >99% cells were viable. We selected 6 AML cell lines as target cells including Kasumi-1 and HL-60 cell lines as the AML M2 type, NB4 as the AML M3 type, K562 as the AML M6 type, HL60/ADR and K562/AO2 cell lines as the chemotherapeutics-resistant AML. First, we evaluated the cytotoxic activity of Vγ9Vδ2 T cells against different AML cell lines. Cytotoxicity was measured by FACS analysis using CFSE and 7-AAD. AML cell lines above were labeled with 1uM CFSE and then incubated with Vγ9Vδ2 T cells for 4h or 8h at the effect: target (E:T) ratios of 5:1, 10:1, and 20:1. 7-AAD was added before acquisition on the FACSCalibur cytometer. The calculation of cytolytic activity was based on the degree of reduction of viable target cells (VTC) with the ability to retain CFSE and exclude 7-AAD (CFSEhigh 7-AAD−). We found Vγ9Vδ2 T cells exerted cytotoxicity on different AML cell lines in varying degrees. At an E:T ratio of 20:1, the cytotoxicity of Vγ9Vδ2 T cells on Kasumi-1, HL-60, HL-60R, NB4, K562, K562-AO2 cell lines for 4h were: 53.27%±18.43% A12.03%±18.16 A13.47%±10.36% A17%±11.01% A29.53%±12.22% A26.4%±0.71%, and for 8h could increase to 56.86%±16.92% A32.25%±6.71% A25.9%±0.99% A41.2%±8.22% A24.27%±8.51% A24.35%±13.51% respectively(P<0.05). Interestingly, Vγ9Vδ2 T cells did not kill normal allogeneic bone marrow mononuclear cells. Then we chose Kasumi-1 cell line, which was the most sensitive to Vγ9Vδ2 T cells' cytotoxicity, as target cells for the study of mechanism below. Kasumi-1 cells were cocultured with 10 folds of Vγ9Vδ2 T cells for 24h and cell morphology was observed by phase contrast microscope. We noted the refractive index of Kasumi-1 cells was significantly decreased, some cells became smaller, shrunken and disintegrated. Also some irregular cells could be seen. It has been proposed that human Vγ9Vδ2 T cells trigger several distinct pathways for killing tumor cells, which include secretion of proinflammatory cytokines and proapoptoticmolecules or cell contact-dependent lysis. Trogocytosis has been reported to play vital roles in killing chronic myeloid leukemia by Vγ9Vδ2 T cells. To know whether Vγ9Vδ2 T cells kill the AML cells in the same way, Vγ9Vδ2 T lymphocytes were further loaded with green-CFSE and coincubated for 4h with Kasumi-1 cells, which were previously labeled with orange-DiI, to measure Vγ9Vδ2 T lymphocytes' ability to conjugate with target cells into lytic synapses by fluorescence microscope. The result revealed that Vγ9Vδ2 T cells were significantly activated after 1h coincubation, and the target cells staying in the middle were surrounded by metatypical Vγ9Vδ2 T cell populations. When the two groups of cells were incubated for 4h, we observed Vγ9Vδ2 T lymphocytes captured the Kasumi-1 cells' membrane fragments by direct contact. In summary, our findings indicate that ex vivo-expanded Vγ9Vδ2 T cells possess a promising cytotoxic activity against not only AML cells, but also chemotherapeutics resistant cells, especially AML M2-type cell line-Kasumi-1, with t (8,21) characteristics. We also discover that the death of target cells relies much upon cell contact with Vγ9Vδ2 T lymphocytes. The further mechanisms involved in Vγ9Vδ2 T cell-mediated cytotoxicity following direct contact and the reason of varying sensitivity still remain unclear. We will clarify all these in the future. Disclosures: No relevant conflicts of interest to declare.
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Gu, Wenbin, Meng Li, Liang Liang, Jian Zhang, Chongye Guo, Zhikai Xing, Yongqing Lan, et al. "Early Different Downstream Target of Glucocorticoid Receptor Contributing to Glucocorticoids Sensitivity in Kasumi-1 Cells." Blood 128, no. 22 (December 2, 2016): 5132. http://dx.doi.org/10.1182/blood.v128.22.5132.5132.
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Abstract The t(8;21) chromosome translocation frequently occurs in acute myeloid leukemia (AML), resulting in an in-frame fusion between the DNA-binding domain of AML1 and almost the entire of ETO gene. The fusion AML1-ETO protein is thought to play a critical role in the abnormal proliferation and differentiation of myeloid leukemia cells, such as Kasumi-1 and SKNO-1 cells. Glucocorticoids (GC) can induce apoptosis in these cells at low concentrations, whereas most other myeloid leukemia cell lines are resistant to glucocorticoid-induced apoptosis. To experimentally address possible sensitive mechanisms in leukemia cells with AML1-ETO translocation, we generated aGC-resistant Kasumi-1 cell line by induction of 10-6 M dexamethasone (Dex) for three weeks. The IC50 of Dex to cells is increased from 2.5×10-8 M for original GC-sensitive Kasumi-1 cell line ( K-S cell line) to more than 1×10-5 M for induced GC-resistant Kasumi-1 cell line (K-R cell line). Since GC resistance often results from mutations in the glucocorticoid receptor (GR), all the exons of GR gene were sequenced and no mutation was found in K-R cells. Comparing to those in K-S cells, the GR protein level didn't decrease in K-R cells after 2h, 4h, 8h, 12h and 24h exposure to dexamethasone. Given that the difference of direct GR downstream genes between K-S and K-R cells may play a key role in the GC sensitivity, we systematically analyzed the changes of gene expression induced by Dex versus ethanol vehicle for 8h in K-S and K-R cells by high throughput RNA sequencing. The time point of 8h was selected according to the expression peaks of several foregone GR target genes after Dex induction. There were found 32 genes conversely regulated in K-S and K-R cells, including 14 mRNAs and 18 long non-coding RNAs. Pathway analysis indicated that the upregulated genes in K-S cells might promote the AML1-ETO fusion protein degradation by proteasomes, while the component genes of this pathway were downregulated in K-R cells. Further validation and function studies of these mRNAs and long non-coding RNAs are ongoing. Our data suggested that the downstream targets of GR among GC-sensitive and -resistant Kasumi-1 cells were significant different and they may contribute to the GC sensitivity and resistance by degradation or reservation of AML-ETO fusion protein and the regulation of apoptosis in t(8;21) leukemia cell subtype. Disclosures No relevant conflicts of interest to declare.
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Douvas, Michael G., L. Roudaiya, J. Grembecka, N. Speck, and J. H. Bushweller. "Development of Allosteric Inhibitors of the Interaction of AML1 and CBFβ for the Treatment of Leukemia." Blood 110, no. 11 (November 16, 2007): 653. http://dx.doi.org/10.1182/blood.v110.11.653.653.
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Abstract The protein-protein interaction between the subunits of the nuclear transcription factor core binding factor, RUNX1 (CBFα) and CBFβ, plays a critical role in hematopoiesis. Chromosomal rearrangements that target CBF genes are among the most common mutations in leukemia, including t(8;21) found in approximately 12% of cases of AML and resulting in the protein AML1-ETO. We describe the development of small molecule inhibitors of the interaction between RUNX1 and CBFβ. We used virtual screening to identify lead compounds. These leads were used to generate a library of compounds to explore the structure-activity relationships and optimize activity, resulting in the identification of low micromolar IC50 inhibitors of this protein-protein interaction. We confirmed by FACS FRET that inhibition also occurs in mammalian cells. HEK-293 cells were transfected with Cerulean-Runt domain and Venus-CBFβ. FRET emission from cells was monitored on a flow cytometer to assess binding of CBFβ and the Runt domain. Results with drug were compared to FRET between Runt and wild-type CBFβ and FRET between Runt and a mutant CBFβ with 2 point mutations abrogating binding to Runt. The small molecule inhibitor KG-3-275 inhibited CBFβ - Runt domain binding in a dose dependent manner (Table 1). Treatment of the t(8;21) leukemia cell lines Kasumi-1 and SKNO-1 results in inhibition of proliferation. SKNO-1 cells are inhibited in a dose-dependent manner by KG-3-275 but not by the weak inhibitor KG-1-253 (Fig 1). KG-3-275 does not inhibit the growth of renal tubular and hepatocellular cell lines (Fig 1). Kasumi-1 and SKNO-1 undergo apoptosis in a dose-dependent manner upon treatment with KG-3-275 (Table 2). Finally, KG-3-275 shows synergy with ATRA in increasing differentiation of Kasumi-1 cells as measured by CD11b expression, consistent with recent published results establishing a link between AML1-ETO and repression of RAR signaling (Fig 2). These data indicate drugs inhibiting CBF interactions hold promise as targeted agents in the treatment of CBF leukemias. Fluorescence Resonance Energy Transfer Geometric Mean in HEK-293 Cells Cerulean-Runt domain + Venus-CBFb 4.07 +/− 0.1 Cerulean-RD + Venus-CBFb61/104 1.85 +/− .09 C-RD + V-CBFb + KG-3-275 200 mcm 3.05 +/− .15 C-RD + V-CBFb + KG-3-275 100 mcm 3.25 +/− 0.1 C-RD + V-CBFb + KG-3-275 50 mcm 3.45 +/− .25 C-RD + V-CBFb + KG-3-275 25 mcm 3.6 +/− 0.2 % Cells Annexin V/PI Negative at 72 Hrs Kasumi-1 0.25% DMSO 91.2 +/− 0.9 KG-3-275 25 mcm 91.2 +/− 1.2 KG-3-275 50 mcm 85.9 +/− 2.4 KG-3-275 100 mcm 66.7 +/− 6.7 SKNO-1 0.25% DMSO 77.6 +/− 3.4 KG-3-275 25 mcm 77.6 +/− 2.3 KG-3-275 50 mcm 63.7 +/− 3.6 KG-3-275 100 mcm 13.7 +/− 6.8 Cellular Proliferation as measured by MTTAssay at 72 Hrs Cellular Proliferation as measured by MTTAssay at 72 Hrs Kasumi-1 Differentiation Measured by CD11b expression at 72 Hrs Kasumi-1 Differentiation Measured by CD11b expression at 72 Hrs
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Ponthan, Frida, Hesta McNeill, Lars Buechler, Vasily Grinev, Josef Vormoor, and Olaf Heidenreich. "Significance of Fusion Genes for Maintenance of Leukaemia." Blood 118, no. 21 (November 18, 2011): 2455. http://dx.doi.org/10.1182/blood.v118.21.2455.2455.
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Abstract Abstract 2455 Background: MLL/AF4 and AML1/MTG8 are fusion genes most frequently found in infant acute lymphoblastic leukaemia (ALL) and acute myeloid leukaemia (AML), respectively. We have previously shown that transient siRNA mediated knock-down of MLL/AF4 and AML/MTG8 impairs proliferation and clonogenicity in vitro and causes a significant increase in median survival in a xeno-transplantation model. Aims: We investigated the role of MLL/AF4 and AML1/MTG8 in leukaemic maintenance and progression of established disease in vivo. We used an inducible lentiviral shRNA expression system to determine the effects of knockdown of MLL/AF4 in the t(4;11)-positive SEM cell line and of AML1/MTG8 in the t(8;21)-positive human leukaemic cell line Kasumi-1. In addition, to allow in vivo imaging SEM and Kasumi-1 cells were labelled with luciferase. Methods: shRNA cassettes specifically targeting the MLL/AF4 and the AML1/MTG8 fusions were cloned into the pTRIPZ vector where shRNA expression and RFP expression are both induced by doxycycline. Luciferase labelled SEM and Kasumi-1 cells were transduced with lentiviral particles, selected with puromycin and transduction efficiency was determined by quantification of RFP positive cells using flow cytometry. Experiments were initiated when more than 65% of the cell populations expressed RFP. Knockdown and expression of known targets of the fusion genes were verified at both the RNA and protein level by qPCR and western blotting, respectively. Cell growth was monitored by cell counts. Immunodeficient NSG mice were given doxycycline in the diet (625ppm) from two days prior to intrafemoral transplantations with 106 luciferase labelled SEM and Kasumi cells transduced with pTRIPZshAML1/MTG8 or pTRIPZshMLL/AF4. The food was changed every other day and disease progression was monitored using in vivo bioluminescence imaging. Results: Upon induction of shRNA expression in vitro Kasumi-1 cells transduced with pTRIPZshAML1/MTG8 showed decreased expression of AML1/MTG8 at protein and RNA levels, which correlated with impaired proliferation. Furthermore, the AML1/MTG8 knockdown resulted in decreased CD34 expression and increased levels of IGFBP7 and PRG2. Induction of shMLL/AF4 expression in SEM cells resulted in decreased expression levels of MLL/AF4 with concomitant decreased expression of HOXA7. However, the number of RFP positive SEM pTRIPZshMLL/AF4 cells decreased over time. When we investigated the in vivo consequences of fusion-gene knockdown in transplanted NSG mice we found no significant differences in overall survival. Notably, mice transplanted with pTRIPZshAML1/MTG8-transduced Kasumi-1 cells showed a lower grade of disseminated disease compared to mice transplanted with pTRIPZshMLL/AF4-transduced Kasumi-1 cells. Furthermore, tumours from these mice had significantly lower RNA and protein levels of AML1/MTG8 (p<0.05). We could not verify knockdown of MLL/AF4 in tumour cells harvested from mice transplanted with SEM pTRIPZshMLL/AF4. Interestingly, these cells showed a complete loss of RFP expression compared to SEM transduced with pTRIPZshAML1/MTG8 (p<0.05). Conclusions: Knockdown of MLL/AF4 and AML1/MTG8 in vitro using an inducible shRNA system led to decreased proliferation and affected genes associated with differentiation. However, the effects were delayed compared to transient siRNA knockdown. In vivo optical imaging is a useful tool to monitor leukaemic progression in vivo. The technique gives information about location and degree of disease dissemination in addition to the overall survival. The loss of RFP expression particularly in SEM pTRIPZshMLL/AF4 cells both in vitro and in vivo highlights the significance of MLL/AF4 in leukaemic maintenance. Further in vitro and in vivo experiments are currently ongoing with knockdown of AML1/MTG8 and MLL/AF4 in alternative cell lines and primary patient-derived material. Disclosures: No relevant conflicts of interest to declare.
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IWATA, T., T. YAGI, and K. KUROSAWA. "Security of the Five-Round KASUMI Type Permutation." IEICE Transactions on Fundamentals of Electronics, Communications and Computer Sciences E91-A, no. 1 (January 1, 2008): 30–38. http://dx.doi.org/10.1093/ietfec/e91-a.1.30.
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Damaj, Issam W. "Higher-Level Hardware Synthesis of the KASUMI Algorithm." Journal of Computer Science and Technology 22, no. 1 (January 2007): 60–70. http://dx.doi.org/10.1007/s11390-007-9007-9.
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Matsui, Hirotaka, Akiko Nagamachi, Yuko Ozaki, Daisuke Aki, Hiroya Asou, Hiroaki Honda, and Toshiya Inaba. "Loss of Titan (Samd9L), a Candidate -7/7q- Responsible Gene Encoding An Actin Remodeling Regulator, Develops MDS/AML in Cooperation with Evi1 or Fbxl10." Blood 114, no. 22 (November 20, 2009): 2963. http://dx.doi.org/10.1182/blood.v114.22.2963.2963.
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Abstract Abstract 2963 Poster Board II-939 From a common microdeletion cluster located in chromosome subband 7q21.3, we identified three candidate responsible genes (Kasumi=Samd9, Titan=Samd9L and Miki=LOC253012) which encode myeloid-tumor suppressors (BBRC 2009). As we presented previously (ASH annual meeting, 2008), Miki encodes a centrosomal protein and is involved in myelodysplasia and chromosomal instability. On the other hand, Kasumi and Titan, that encode 60% identical proteins, are poorly characterized. Recent reports revealed that bi-allelic point mutations in Kasumi gene cause a rare fatal skin disease, Normophosphatemic Familial Tumoral Calcinosis (NFTC). To identify the contributions of these genes to leukemogenesis, we initially generated Titan deficient mice (mouse genome contains only Titan and lacks Kasumi gene). Although heterozygous (titan+/−) and homozygous (titan-/-) mice were born and grown normally without hematological abnormalities, they naturally developed AML at high frequency after they reached 20 months old. This unusually long latency suggests that additional gene alterations are required for leukemia development. Thus we attempted to accelerate the onset of leukemia by retroviral insertional mutagenesis. Virus infection induced various myeloid leukemias after 10 to 12 months in almost all titan+/− and titan-/- mice. Inverse PCR detected two common virus integration sites specific for titan+/− and titan-/- mice, which induced deregulated expression of a zinc finger transcription factor, Evi1, and a histone H3K36 demethylase, Fbxl10. We next performed mouse BMT using titan-/- and +/+ bone marrow cells transduced with Evi1 retrovirally. Mice transplanted with titan(+/+)/Evi1 overexpressing cells developed MDS or AML after 7 months. By contrast, most mice transplanted with titan(-/-)/Evi1 overexpressing cells developed AML within 6 months after BMT, confirming co-operation between loss of Titan and Evi1 overexpression in myeloid leukemogenesis. Both Evi1 and Fbxl10 are reported to downregulate p15Ink4b tumor suppressor gene. Moreover, in human secondary MDS, DNA methylation in the promoter region of p15Ink4b is closely associated with 7q deletion. Thus we compared Fbxl10 and p15Ink4b expressions between AML/MDS samples with or without 7q deletion. We found that 7q deletion was correlated with higher Fbxl10 and with lower p15Ink4b levels, suggesting that silencing of p15ink4b through transcriptional and epigenetical mechanisms would be involved in leukemia with 7q deletion. To elucidate the function of Kasumi and Titan, we firstly immunoprecipitated Titan binding proteins from FLAG-Titan expressing cells and identified two specific bands around 150 and 70KDa. Mass spectrometry analysis showed that they correspond to Flightless1 (Fli1) and Scinderin (Scin), respectively. Because both Fli1 and Scin belong to the gelsolin superfamily proteins that bind to and sever actin filaments, we speculated that Titan is involved in cell movement via actin remodeling. To analyze this, we established Titan-knockdown (K/D) mouse fibloblasts by introducing Titan-specific shRNA-expressing vectors and observed their migration under time-lapse microscopy. In wound-healing assay, Titan-K/D cells migrated slower towards wound edge with loss of polarity. Each cell moved restlessly by quickly changing the direction. In these cells, lamellipodial protrusions rapidly formed and retracted. In accordance with this phenotype, activity of Rac1, a Rho GTPase, increased in Titan-K/D cells. This abnormality in cell migration is likely involved in pathogenesis of NFTC, i.e., severe inflammations in skin and mucosae. Moreover, since recent reports revealed the contribution of hyperactivated Rac1 to transformation of hematopoietic stem cells through abnormal actin remodeling, our findings suggest that deletion of Kasumi or Titan is a cue to cause AML/MDS through aberrant Rac1 activation. Disclosures: No relevant conflicts of interest to declare.
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Velthaus, Arne, Kerstin Cornils, Saskia Grüb, Hauke Stamm, Daniel Wicklein, Carsten Bokemeyer, Michael Heuser, Sabine Windhorst, Walter Fiedler, and Jasmin Wellbrock. "The Actin Binding Protein Plastin-3 Is Involved in the Pathogenesis of Acute Myeloid Leukemia." Blood 128, no. 22 (December 2, 2016): 1662. http://dx.doi.org/10.1182/blood.v128.22.1662.1662.
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Abstract Leukemia-initiating cells reside within the bone marrow (BM) in specialized niches where they undergo complex interactions with their surrounding stromal cells. In order to identify genes being implicated in the interaction of acute myeloid leukemia (AML) cells and stromal cells, we performed co-cultures of primary AML cells with primary endothelial cells and osteoblasts. The gene expression of co-cultured AML blasts was compared to AML cells grown without adherent cells using microarray analysis. Amongst those genes being dysregulated upon co-culture was the actin binding protein plastin-3 (PLS3). Further RT-qPCR analysis revealed an endogenous PLS3 expression in about 50% of BM samples from AML patients (n=25). In contrast, expression of PLS3 was only detected in 2 of 12 analyzed AML cell lines with Kasumi-1 showing strong and THP-1 showing only weak expression. Therefore, functional analysis of PLS3 in AML was studied using shRNA knockdown and overexpression of PLS3 in Kasumi-1 cells. We could show that PLS3 has an impact on the colony formation capacity of AML cells in vitro as the knockdown resulted in significantly reduced colony numbers while increased colony growth was observed in the Kasumi-1 cells overexpressing PLS3 (p<0.001 and p<0.001, respectively). To investigate the role of PLS3 in vivo, NSG mice were transplanted with the PLS3 knockdown Kasumi-1 cells. Compared to mice transplanted with Kasumi-1 cells transduced with a vector carrying a scrambled shRNA, the PLS3 knockdown mice survived significantly longer (median survival time 64 vs. 110 days, respectively; p<0.001; n=9 mice per group). Finally, we investigated whether the expression of PLS3 was associated with AML patients' outcome using published microarray-based gene expression data (Verhaak et al, Haematologica 2009;94). Clinical data of 290 AML patients were available. Based on the mean gene expression value, the patient cohort was divided into high vs low PLS3 expressors. The overall survival was analyzed in a multivariate Cox proportional hazards model including PLS3 gene expression and the baseline parameters age, karyotype and FLT3 mutational status. After a stepwise removal of insignificant terms, the patient's age and a high PLS3 expression remained as independent prognostic survival markers (for PLS3: HR 1.58 (CI 1.05 - 2.37) and for age: HR 1.01 (CI 1.00 - 1.03)). In conclusion, our results identify the actin binding protein PLS3 as potential novel therapeutic target in AML. Disclosures Stamm: Astellas: Other: Travel, Accommodation, Expenses. Heuser:BerGenBio: Research Funding; Tetralogic: Research Funding; Novartis: Consultancy, Research Funding; Celgene: Honoraria; Bayer Pharma AG: Research Funding; Pfizer: Research Funding; Karyopharm Therapeutics Inc: Research Funding. Fiedler:Kolltan: Research Funding; Ariad/Incyte: Consultancy; Novartis: Consultancy; Gilead: Other: Travel; Teva: Other: Travel; GSO: Other: Travel; Pfizer: Research Funding; Amgen: Consultancy, Other: Travel, Patents & Royalties, Research Funding. Wellbrock:Astellas: Other: Travel, Accommodation, Expenses.
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Mahmud, Hasan, Frank JG Scherpen, Tiny Meeuwsen-de Boer, Harm Jan Lourens, and Eveline S. de Bont. "Kinome Profiling Identifies Phospholipase C Gamma 1 (PLCγ1) As a Potential Target for t(8;21) AML." Blood 126, no. 23 (December 3, 2015): 1384. http://dx.doi.org/10.1182/blood.v126.23.1384.1384.
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Abstract The t(8;21) (q22;q22) chromosomal translocation is one of the most frequent genetic alterations in acute myeloid leukemia (AML). Still about 30% of patients with t(8;21) AML struggle with relapse, despite intensive chemotherapy. We and others have shown that kinase activity profiling is able to identify new potential druggable targets. The aim of this study was to perform kinome profiling on 29 AML patients (cytogenetically normal, CN-AML, n=17 and t(8;21), n=12) and 4 normal bone marrow (NBM) samples to identify new potential druggable targets relevant to t(8;21) AML. We defined 130 peptides differentially phosphorylated between CN-AML, t(8;21) and NBM (p<0.05). With this technique, phospholipase C gamma 1 (PLCγ1_Y783) was identified as one of the highest phosphorylated peptide in t(8;21) AML. The knowledge about the role of PLCγ1 in leukemia progression is unknown. Therefore, we selected PLCγ1 to study its functional role in t(8;21) AML cell line (Kasumi-1) and also to test its efficacy as a potential target and provide proof of principle for this approach. PLCγ1 peptide phosphorylation was significantly higher in all t(8;21) AML samples when compared to NBM or to CN-AML (p<0.001 and p<0.05 respectively). mRNA expression of PLCγ1 in a publicly available paediatric AML database (http://r2.amc.nl) also showed significantly higher expression in t(8;21) AML compared to other AML karyotypes (p<0.001). PLCγ1 silencing with the specific short hairpin RNAs (two constructs, shRNAs PLCγ1-A and shRNAs PLCγ1-B), showed 50% and 80% decrease in PLCγ1 mRNA level compared with the control (scramble shRNA). These results were confirmed by PLCγ1 protein level analysis by western blotting. The shRNA-mediated silencing of PLCγ1 leads to suppression of the Kasumi-1 cell proliferation and viability at day 8 after transduction (p<0.05). The percentage of apoptosis in PLCγ1 supressing kasumi-1 cells at day 4 was two-fold higher than in scramble (p<0.01). To understand mechanistic insights of inhibited cell proliferation of PLCγ1 knock down cells, human phosphokinase microarray was performed and higher phosphorylation of Chk2_T68 and AMPK-α1_T183 were found in these cells. Higher Chk2 phosphorylation may responsible for cell cycle arrest and induced cell death of PLCγ1 supressing kasumi-1 cells. Similarly, higher phosphorylation of AMPK-α1 may inhibited cell proliferation of PLCγ1 knockdown cells. In conclusion kinome profiling is an elegant approach to identify interesting potential druggable targets. The results from proof of principle experiments strongly support the notion that suppression of PLCγ1 inhibited cell growth and induced apoptosis in Kasumi-1 cells. Disclosures No relevant conflicts of interest to declare.
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Tabe, Yoko, Rooha Contractor, Susanne Radke, Michael Andreeff, and Marina Konopleva. "Histone Deacetylase Inhibitor Depsipeptide (FK228) Induces Apoptosis through Histone Acetylation and Upregulation of Annexin A1 in AML1-ETO Expressing Kasumi-1 Cells." Blood 104, no. 11 (November 16, 2004): 4343. http://dx.doi.org/10.1182/blood.v104.11.4343.4343.
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Abstract The t(8;21) translocation, found in 12% of AML, creates the chimeric fusion protein, AML-ETO, which recruits the HDAC complex to AML-1-dependent promoters resulting in transcription repression. Here, we studied the effects of HDAC inhibitor FK228 in the AML1-ETO positive AML cell line, Kasumi-1 and investigated molecular mechanisms of apoptosis induction in these cells. FK228 (5nM, 24 hours) caused marked growth inhibition, apoptosis induction (annexinV+/PI-; control 4.5±0.5%, FK228 24.6±2.6%), and cell cycle arrest (G1 phase; control 59.5%, FK228 76.7%, S phase; control 32.1%, FK228 17.8%). To investigate the molecular changes induced by FK228 we used cDNA array technology. After 24 hour exposure of Kasumi-1 cells, FK228 down-regulated (≤2-fold) 14 genes and up-regulated (≥2-fold) 123 genes including apoptosis and cell cycle regulating genes (Annexin A1; 3.5 fold, serglycin; 2.6 fold, Hes1; 2.2 fold, and TNFα; 2.1 fold increase). Among these genes, we focused on the pro-apoptotic protein Annexin A1, one of the calcium-dependent phospholipid binding proteins. First, we confirmed induction of Annexin A1 mRNA by FK228 using quantitative TaqMan RT-PCR (7.2±1.7 fold). As demonstrated by Western blot analysis, FK228 upregulated the expression of both, full-length Annexin A1 protein and a cleaved isoform of Annexin A1. To identify the critical histone residue modified by FK228 on the Annexin A1 promoter, we performed chromatin immunoprecipitation (ChIP) assays quantified by TaqMan PCR. FK228 increased H4 acetylation (6.7 ±1.8 fold) and H3-K9 acetylation (3.8 ±0.4 fold compared with control at 24 hours) in the Annexin A1 promoter. Next, we investigated effects of FK228 on apoptosis and signal transduction pathways that may modulate Annexin A1 function. As cleavage of the ubiquitous protein Annexin A1 is reported to be associated with caspase activation, the effect of caspase3 inhibitor Z-DEVD-cfm was examined. Z-DEVD pretreatment of Kasumi-1 cells resulted in increased viability and partial inhibition of apoptosis by FK228 (45% decrease in apoptosis with 20μM Z-DEVD). Z-DEVD partially inhibited FK228 induced Annexin A1 cleavage isoform indicating that cleavage of Annexin A1 involves both, caspase-dependent and independent mechanisms. Since activation of the MAPK/ERK pathway has recently been linked to histone de-acetylation, we examined the effects of FK228 on ERK phosphorylation. FK228 used at 5nM inhibited ERK signaling. Further experiments are ongoing to elucidate the possible association of MAPK inhibition, histone acetylation and Annexin A1 gene expression in Kasumi-1 cells. In summary, these findings suggest that FK228 induces apoptosis and growth inhibition in Kasumi-1 cells in part by inducing Annexin A1 expression through histone acetylation of the promoter in association with caspase-dependent Annexin 1 cleavage and inhibition of MAPK signaling.
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Asou, H., S. Tashiro, K. Hamamoto, A. Otsuji, K. Kita, and N. Kamada. "Establishment of a human acute myeloid leukemia cell line (Kasumi-1) with 8;21 chromosome translocation." Blood 77, no. 9 (May 1, 1991): 2031–36. http://dx.doi.org/10.1182/blood.v77.9.2031.2031.
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Abstract A novel leukemic cell line with an 8;21 chromosome translocation, designated as Kasumi-1, was established from the peripheral blood of a 7-year-old boy suffering from acute myeloid leukemia (AML). The Kasumi- 1 cells were positive for myeloperoxidase showing a morphology of myeloid maturation. The response in proliferation assay was observed in the culture with interleukin-3 (IL-3), IL-6, granulocyte colony- stimulating factor (G-CSF), and granulocytemacrophage CSF (GM-CSF), but not with IL-1 or IL-5. Neither granulocytic nor eosinophilic maturation was observed in the liquid culture by the addition of dimethyl sulfoxide, G-CSF, or IL-5, respectively. In contrast, induction of macrophagelike cells was seen by the addition of phorbol ester. This is the first report of a human AML cell line with t(8;21) that has characteristics of myeloid and macrophage lineages. The cell line could be a useful tool for elucidating the pathophysiology of AML with t(8;21).
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Asou, H., S. Tashiro, K. Hamamoto, A. Otsuji, K. Kita, and N. Kamada. "Establishment of a human acute myeloid leukemia cell line (Kasumi-1) with 8;21 chromosome translocation." Blood 77, no. 9 (May 1, 1991): 2031–36. http://dx.doi.org/10.1182/blood.v77.9.2031.bloodjournal7792031.
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A novel leukemic cell line with an 8;21 chromosome translocation, designated as Kasumi-1, was established from the peripheral blood of a 7-year-old boy suffering from acute myeloid leukemia (AML). The Kasumi- 1 cells were positive for myeloperoxidase showing a morphology of myeloid maturation. The response in proliferation assay was observed in the culture with interleukin-3 (IL-3), IL-6, granulocyte colony- stimulating factor (G-CSF), and granulocytemacrophage CSF (GM-CSF), but not with IL-1 or IL-5. Neither granulocytic nor eosinophilic maturation was observed in the liquid culture by the addition of dimethyl sulfoxide, G-CSF, or IL-5, respectively. In contrast, induction of macrophagelike cells was seen by the addition of phorbol ester. This is the first report of a human AML cell line with t(8;21) that has characteristics of myeloid and macrophage lineages. The cell line could be a useful tool for elucidating the pathophysiology of AML with t(8;21).
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Duque-Afonso, Jesus, Tobias Berg, Olaf Heidenreich, and Michael Luebbert. "Epigenetic Repression of the Adaptor Molecule LAT2 by the Leukemic Fusion Protein AML1/ETO." Blood 110, no. 11 (November 16, 2007): 987. http://dx.doi.org/10.1182/blood.v110.11.987.987.
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Abstract The chromosomal translocation (8;21) fuses the hematopoietic transcription factor AML1 (RUNX1) with ETO, resulting in the leukemia-specific chimeric protein AML1/ETO. This fusion protein represses transcription by recruiting a nuclear co-repressor complex containing HDACs and DNMT1 to its target promoters. Previously, we have identified a novel in vivo AML1/ETO target gene, LAT2 (NTAL/LAB/WBSCR5), which is involved in FcεR I, c-Kit, B cell- and T cell receptor signalling. Notably, LAT2 is strongly repressed in AML1/ETO positive cells including primary AML blasts, which was confirmed by others in several large AML cohorts. We have now addressed the molecular mechanisms of AML1/ETO-mediated LAT2 repression. AML1/ETO was induced by Ponasterone A in an ecdysone-inducible system in U937 cells (9/14/18 cell line). To deplete AML1/ETO in t(8;21)-positive cells, we electroporated Kasumi-1 cells with AML1/ETO siRNA. To interfere with epigenetic modifications more directly, cells were treated with the DNMT inhibitor decitabine (DAC) and 4 different HDAC inhibitors. LAT2 expression was determined by Northern Blot, qRT-PCR and Western Blot. HDAC occupation and the histone status of the LAT2 promoter was examined by chromatin immunoprecipitation (ChIP). LAT2 mRNA was downregulated already after 4 hours of conditional expression of AML1/ETO in 9/14/18 cells, and constitutively repressed in the AML1/ETO-positive Kasumi-1 and SKNO-1 cells. siRNA-mediated AML1/ETO depletion caused a 9-fold upregulation of LAT2 in Kasumi-1 cells, suggesting a possible direct mechanism of repression. To address this question, we performed ChIP assays for the LAT2 promoter after AML1/ETO induction in 9/14/18 cells. AML1/ETO inhibited acetylation of histone H3, H3K9 and H4, but did not affect trimethylation of H3K4. These changes were associated with the recruitment of HDAC2, but not HDAC1 and HDAC3, to the LAT2 promoter. The HDAC inhibitors MS-275, SAHA, TSA and valproic acid induced LAT2 mRNA in a dose-dependent manner in AML1/ETO-expressing Kasumi-1, with MS-275 being the most efficient inhibitor. MS-275 induced LAT2 expression also in t(8;21)-positive SKNO-1, but not in AML1/ETO-negative HL60 and U937 cells. LAT2 mRNA was also upregulated in a dose-dependent manner after DAC treatment in Kasumi-1 cells. The combination of DAC and MS-275 had a synergistic effect on inhibition of cell growth, acetylation of histones H3 and H4, and re-expression of LAT2 mRNA. MS-275-mediated re-expression of LAT2 was associated with an increase in acetylation of histone H3, H3K9, H4 and trimethylation of H3K4. The increase of activating histone modifications was associated with the release of HDAC1, HDAC2 and HDAC3 from the LAT2 promoter. In conclusion, the epigenetic changes of the LAT2 promoter caused by AML1/ETO could be pharmacologically reverted by inhibition of histone acetylation.
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Kang, Ju-Sung, Bart Preneel, Heuisu Ryu, Kyo Il Chung, and Chee Hang Park. "Pseudorandomness of Basic Structures in the Block Cipher KASUMI." ETRI Journal 25, no. 2 (April 15, 2003): 89–100. http://dx.doi.org/10.4218/etrij.03.0102.0210.
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Choi, Hyun-Jun, Young-Ho Seo, Sung-Sik Moon, and Dong-Wook Kim. "Hardware Design and Application of Block-cipher Algorithm KASUMI." Journal of the Korean Institute of Information and Communication Engineering 15, no. 1 (January 31, 2011): 63–70. http://dx.doi.org/10.6109/jkiice.2011.15.1.063.
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Yasir, Ning Wu, Zain Anwar Ali, Muhammad Mujtaba Shaikh, Muhammad Rehan Yahya, and Muhammad Aamir. "Compact and High Speed Architectures of KASUMI Block Cipher." Wireless Personal Communications 106, no. 4 (February 22, 2018): 1787–800. http://dx.doi.org/10.1007/s11277-018-5606-8.
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Madani, Mahdi, and Camel Tanougast. "FPGA implementation of an enhanced chaotic-KASUMI block cipher." Microprocessors and Microsystems 80 (February 2021): 103644. http://dx.doi.org/10.1016/j.micpro.2020.103644.
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ZHAO, Xue, and Shu-xu GUO. "High-Performance Hardware Implementation of the 3GPP Algorithm KASUMI." Journal of China Universities of Posts and Telecommunications 13, no. 1 (March 2006): 60–62. http://dx.doi.org/10.1016/s1005-8885(07)60082-x.
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Muthalagu, Raja, and Subeen Jain. "Improved KASUMI block cipher for GSM-based mobile networks." Journal of Cyber Security Technology 4, no. 4 (July 23, 2020): 197–210. http://dx.doi.org/10.1080/23742917.2020.1796252.
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SUGIO, N., H. AONO, S. HONGO, and T. KANEKO. "A Study on Higher Order Differential Attack of KASUMI." IEICE Transactions on Fundamentals of Electronics, Communications and Computer Sciences E90-A, no. 1 (January 1, 2007): 14–21. http://dx.doi.org/10.1093/ietfec/e90-a.1.14.
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Mitkevich, Vladimir A., Olga V. Kretova, Irina Yu Petrushanko, Ksenia M. Burnysheva, Dmitry V. Sosin, Olga V. Simonenko, Olga N. Ilinskaya, Nickolai A. Tchurikov, and Alexander A. Makarov. "Ribonuclease binase apoptotic signature in leukemic Kasumi-1 cells." Biochimie 95, no. 6 (June 2013): 1344–49. http://dx.doi.org/10.1016/j.biochi.2013.02.016.
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