Apoptosis In Kasumi-1 Cells Research Articles

GRK2 Protein Mediates the ANRIL, a lncRNA, to Affect the Proliferation and Apoptosis of Kasumi-1 Cells.

A long non-coding RNAs (LncRNAs) called antisense noncoding RNA in the INK4 locus (ANRIL), has emerged as substantial regulators of cell survival in acute myeloid leukemia (AML). However, its speciffc and potential mechanism is uncertain in AML. In this research, we investigated the role of ANRIL in cell proliferation, apoptosis, and the underlying mechanism in AML cells. ANRIL expression was quantified by real-time quantitative polymerase chain reaction (RT-qPCR). Kasumi-1 cells were transfected with LV-ANRIL plasmid to upregulate ANRIL expression, with or without co-transfection with a G Protein-Coupled Receptor Kinase 2 (GRK2) siRNA. Additionally, these cells were transfected with sh-ANRIL plasmid to inhibit ANRIL expression, with or without co-transfection with a GRK2 overexpression plasmid. Cell proliferation and apoptosis were determined using the cell counting kit-8 (CCK8) and flow cytometry. Protein expression levels of phosphatidylinositide 3-kinases (PI3K), protein kinase B (AKT), phosphorylated-Akt (p-AKT), Bcl-2-associated protein x (BAX), B-cell leukemia/lymphoma 2 protein (BCL-2), proliferating cell nuclear antigen (PCNA), cleaved caspase-3, and GRK2 were detected by western blot. The RNA-binding protein immunoprecipitation (RIP) assay was conducted to investigate the interaction between ANRIL and GRK2. ANRIL expression was increased in Kasumi-1 cells. ANRIL upregulation expression promoted cell proliferation and inhibited apoptosis. Furthermore, its upregulation led to increased expressions of PI3K, AKT, p-AKT, PCNA, and BCL-2, and decreased expression of BAX in Kasumi-1 cells. Additionally, transfection with GRK2 siRNA attenuated the promoting effect of LV-ANRIL on Kasumi-1 cells proliferation and the PI3K/AKT pathway, increased BAX and cleaved caspase-3 expressions, and decreased BCL-2 and PCNA expressions. GRK2 overexpression reversed the inhibitory effect of sh-ANRIL on cell proliferation and the PI3K/AKT pathway. Furthermore, it promoted BCL-2 and PCNA expressions, and inhibited BAX and cleaved caspase-3 expressions. RIP assay confirmed the physical interaction between ANRIL and GRK2. The GRK2 protein-mediated ANRIL, increasing Kasumi-1 cell proliferation and inhibiting apoptosis by activating the PI3K/AKT/BCL-2 pathway.

Effect of Selinexor on Proliferation and Apoptosis of Acute Myeloid Leukemia Kasumi-1 Cells

To investigate the effects of selinexor, a inhibitor of nuclear export protein 1 (XPO1) on the proliferation inhibition and apoptosis of Kasumi-1 cells in acute myeloid leukemia (AML). MTS method was used to detect the inhibitory effect of different concentrations of selinexor on the proliferation of Kasumi-1 cells at different time points. The apoptosis rate and cell cycle changes after treatment with different concentration of selinexor were detected by flow cytometry. Selinexor inhibited the growth of Kasumi-1 cells at different time points in a concentration-dependent manner (r 24 h=0.7592, r 48 h=0.9456, and r 72 h=0.9425). Selinexor inhibited Kasumi-1 cells growth in a time-dependent manner (r =0.9057 in 2.5 μmol/L group, r =0.9897 in 5 μmol/L group and r =0.9994 in 10 μmol/L group). Selinexor could induce apoptosis of Kasumi-1 cells in a dose-dependent manner (r =0.9732), and the apoptosis of Kasumi-1 cells was more obvious with the increase of drug concentration. The proportion of G0/G1 phase was significantly increased and the proportion of S phase was significantly decreased after the treatment of Kasumi-1 cells by selinexor. With the increase of drug concentration, the proportion of Kasumi-1 cells cycle arrest in G0/G1 phase was increased and the cell synthesis was decreased. Selinexor can promote the death of tumor cells by inhibiting Kasumi-1 cells proliferation, inducing apoptosis and blocking cell cycle.

SPHINX-Based Combination Therapy as a Potential Novel Treatment Strategy for Acute Myeloid Leukaemia.

Introduction: Dysregulated alternative splicing is a prominent feature of cancer. The inhibition and knockdown of the SR splice factor kinase SRPK1 reduces tumour growth in vivo. As a result several SPRK1 inhibitors are in development including SPHINX, a 3-(trifluoromethyl)anilide scaffold. The objective of this study was to treat two leukaemic cell lines with SPHINX in combination with the established cancer drugs azacitidine and imatinib. Materials and Methods: We selected two representative cell lines; Kasumi-1, acute myeloid leukaemia, and K562, BCR-ABL positive chronic myeloid leukaemia. Cells were treated with SPHINX concentrations up to 10μM, and in combination with azacitidine (up to 1.5μg/ml, Kasumi-1 cells) and imatinib (up to 20μg/ml, K562 cells). Cell viability was determined by counting the proportion of live cells and those undergoing apoptosis through the detection of activated caspase 3/7. SRPK1 was knocked down with siRNA to confirm SPHINX results. Results: The effects of SPHINX were first confirmed by observing reduced levels of phosphorylated SR proteins. SPHINX significantly reduced cell viability and increased apoptosis in Kasumi-1 cells, but less prominently in K562 cells. Knockdown of SRPK1 by RNA interference similarly reduced cell viability. Combining SPHINX with azacitidine augmented the effect of azacitidine in Kasumi-1 cells. In conclusion, SPHINX reduces cell viability and increases apoptosis in the acute myeloid leukaemia cell line Kasumi-1, but less convincingly in the chronic myeloid leukaemia cell line K562. Conclusion: We suggest that specific types of leukaemia may present an opportunity for the development of SRPK1-targeted therapies to be used in combination with established chemotherapeutic drugs.

Efficient delivery of the Bcl-2 antisense oligonucleotide G3139 via nucleus-targeted aCD33-NKSN nanoparticles

G3139 is an antisense oligodeoxyribonucleotide (ODN) developed as a Bcl-2 down-regulating agent for the treatment of acute myelogenous leukemia (AML). However, the clinical efficacy of G3139 has been shown to be limited due to its rapid plasma clearance and low permeability. To enhance the effective delivery of G3139, this work prepared a novel nano gene delivery vector (aCD33-NKSN) consisting of a CD33 antigbody (aCD33), a nuclear localization signal (NLS), gene fusion peptides (KALA), and stearic acid (SA) for CD33 antigen targeting and nuclear localization. The aCD33-NKSN/G3139 nanoparticles were spherical and uniformly sized with a positive charge and sustained release. They had an excellent G3139 loading capacity and colloidal stability. The aCD33-NKSN/G3139 delivered G3139 into the nucleus of Kasumi-1 cells and aCD33-NKSN/G3139 could more effectively inhibited Bcl-2 expression and induced apoptosis in Kasumi-1 cells versus free G3139. The aCD33-NKSN/G3139 administration was more effective at inhibiting tumor growth, and significantly prolonged the survival time of mice in contrast to free G3139. The results illustrate that aCD33-NKSN/G3139 nanoparticles could improve the antitumor activity of encapsulated G3139 due to aCD33 targeting and the ability to perform nuclear localization, The results offer a promising clinical application potential for the treatment of acute myeloid leukemia.

Effect of Down-Regulation of ANRIL on Proliferation and Apoptosis of Kasumi-1 Cells and Its Potential Mechanism

To investigate the down-regulation of ANRIL (Antisense non-coding RNA in the INK4 Locus) effects on proliferation and apoptosis of Kasumi-1 cells and its related molecular mechanism. Recombinant lentivirus was used to construct ANRIL down-regulation Kasumi-1 cells (sh-ANRIL group) and its control cells (sh-NC group). A fluorescence microscope was used to observe the transfection efficiency, RT-qPCR was used to detect knockdown efficiency and ANRIL expression in PBMCs and MBMCs of patients with acute myeloid leukemia (AML). Proliferation and apoptosis of Kasumi-1 cells were assayed by CCK-8 method and flow cytometry. Western blot was employed to detect the expression of PI3K, AKT, p-AKT, and relevant protein after down-regulation of ANRIL in Kasumi-1 cells. ANRIL overexpressed significantly in PBMCs and MBMCs of patients with AML, the transfection efficiency of recombinant lentivirus carrying sh-ANRIL and sh-NC on Kasumi-1 cells exceeded 90%, and the knockdown efficiency was 70%. When DNR was administrated for 24, 48, and 72 hours, the cell inhibition rate of the sh-ANRIL group was (47.40±1.49)%, (69.11±0.51)% and (91.82±1.10)%, which were significantly higher than those of the sh-NC group, respectively (P<0.05). The apoptotic rate in the sh-ANRIL group was (10.29±0.58)%, which was significantly higher than (5.42±0.67)% of the sh-NC group (P<0.01). After DNR treatment for 24 hours, the apoptotic rate of the sh-ANRIL group was (54.41±1.69)%, which was significantly higher than (38.28±1.42)% of sh-NC group (P<0.001). Western blot revealed that the protein levels of PI3K, p-AKT, PCNA, and BCL-2 in the sh-ANRIL group were reduced significantly than those in the sh-NC group, while the BAX protein expression increased. ANRIL may affect the proliferation and apoptosis of Kasumi-1 cells through PI3K/AKT signaling pathway. ANRIL is a potential therapeutic target for AML.

The Cytotoxic Effects of Moringa oleifera Leaf Extract and Silver Nanoparticles on Human Kasumi-1 Cells.

BackgroundMoringa oleifera, commonly known as “moringa”, is widely cultivated in tropical and subtropical regions across the globe. Extensive studies have shown that various parts of the moringa tree exhibit anti-cancer properties. This study determined the effects of sequential moringa leaf extracts and silver nanoparticles synthesized from moringa leaf extract on Kasumi-1 leukemia cells.Methods and ResultsDried moringa leaf powder was sequentially extracted with the assistance of ultrasound starting with absolute ethanol, followed by 50% ethanol, and finally, deionized water. The aqueous extract was utilized to synthesize silver nanoparticles. The optimum conditions to generate moringa silver nanoparticles (MO-AgNPs) were eight hours of incubation at 60°C with 1 mM silver nitrate and 1% moringa aqueous extract from sequential extraction. The three extracts and MO-AgNPs were used to treat Kasumi-1 cells for 24, 48, 72 hours with concentrations ranging from 400 to 12.5 µg/mL, while cell viability was determined with 3(4, 5-dimethythiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. After 72 hours of treatment, the moringa leaf absolute ethanol extract displayed the strongest inhibitory effects on Kasumi-1 cells with IC50 of 10 µg/mL, in comparison to moringa leaf 50% ethanol extract (25 µg/mL) and aqueous extract (>400 µg/mL). Interestingly, MO-AgNPs exhibited the strongest cytotoxic effects on Kasumi-1 cells with an IC50 of 7.5 µg/mL. Cytotoxic study on normal CD34+ cells treated with up to 50ug/mL of either MO-AgNPs or ethanol extract still had more than 80% cell viability indicating that the treatments have selective cytotoxicity against the cancer cells. Morphological studies of Kasumi-1 cells treated with IC50 of moringa leaf ethanolic extract and MO-AgNPs show a lot of shrinking, dying cells and cell debris. Cell cycle studies displayed an increase in cells at the G1 phase for ethanol leaf extract, while MO-AgNPs caused cell cycle arrest at the S phase after treatment with IC50 dose for 24 hours. Moringa leaf ethanol extract and the nanoparticles induced apoptosis in Kasumi-1 cells as shown by annexin V – FITC assays. Gene expression analysis by qPCR verified these outcomes, as the moringa leaf ethanol extract led to significant upregulation of proapoptotic gene caspase 8, whereas the MO-AgNPs caused a significant increase of proapoptotic protein BID.ConclusionThis study reveals that moringa ethanolic leaf extract and MO-AgNPs induced potent antiproliferative effects in Kasumi-1 cells by apoptosis.

Stable depletion of RUNX1-ETO in Kasumi-1 cells induces expression and enhanced proteolytic activity of Cathepsin G and Neutrophil Elastase.

The oncogenic fusion protein RUNX1-ETO is a product of the t(8;21) translocation and consists of the hematopoietic transcriptional master regulator RUNX1 and the repressor ETO. RUNX1-ETO is found in 10–15% of acute myeloid leukemia and interferes with the expression of genes that are essential for myeloid differentiation. The neutrophil serine protease Cathepsin G is one of the genes suppressed by RUNX1-ETO, but little is known about its impact on the regulation of other lysosomal proteases. By lentiviral transduction of the t(8;21) positive cell line Kasumi-1 with an RUNX1-ETO specific shRNA, we analyzed long-term effects of stable RUNX1-ETO silencing on cellular phenotypes and target gene expression. Stable anti RUNX1-ETO RNAi reduces both proliferation and apoptosis in Kasumi-1 cells. In addition, long-term knockdown of RUNX1-ETO leads to an upregulation of proteolytic activity in Kasumi-1 cells, which may be released in vitro upon cell lysis leading to massive degradation of cellular proteins. We therefore propose that protein expression data of RUNX1-ETO-silenced Kasumi-1 cells must be analyzed with caution, as cell lysis conditions can heavily influence the results of studies on protein expression. Next, a mass spectrometry-based approach was used to identify protease cleavage patterns in RUNX1-ETO-depleted Kasumi-1 cells and Neutrophil Elastase has been identified as a RUNX1-ETO candidate target. Finally, proteolytic activity of Neutrophil Elastase and Cathepsin G was functionally confirmed by si/shRNA-mediated knockdown in Kasumi-1 cells.

PI3 Kinase p110 Delta Is Required for Leukemic Cell Survival and Self-Renewal in t(8;21) Acute Myeloid Leukemia

Acute myeloid leukemia (AML) with t(8;21) comprises about 10% of adult AML cases, and is the most frequent genetic alteration in pediatric AML. In t(8;21) AML, the AML1-ETO transcription factor fusion dysregulates transcription in hematopoietic cells, interfering with apoptosis and differentiation, and leading to enhanced self-renewal. The phosphoinositide-3-kinase (PI3K) signaling pathway is frequently overactivated in AML, representing a promising target in AML. However, PI3K also plays important roles in normal hematopoiesis. Hematopoietic cells express four catalytic subunits of Class I PI3K (p110α, p110β, p110γ, and p110δ). Previous studies have demonstrated that these isoforms have redundant roles in normal hematopoietic stem cells (HSCs), but play specific roles in some genetic subtypes of AML. Therefore, inhibiting individual PI3K isoforms in AML may allow for therapeutic targeting of this pathway with lower toxicity. In fact, isoform-selective PI3K inhibitors are already in clinical development, and the p110δ-selective inhibitor idelalisib is approved by the FDA for lymphoid malignancies.To determine whether t(8;21) AML cells are dependent upon specific PI3K isoforms, we performed proliferation and apoptosis assays in Kasumi-1 cells, a t(8;21) human AML cell line, with PI3K inhibitors: pictilisib (GDC0941, a pan-PI3K inhibitor), buparlisib (BKM120, a pan-PI3K inhibitor), alpelisib (BYL719, a p110α-selective inhibitor), idelalisib (GS1101,a p110δ-selective isoform inhibitor) and KIN193 or TGX221 (p110β-selective inhibitors). We observed a decrease in proliferation and in Akt/mTOR signaling with the pan-PI3K inhibitors and alpelisib. The pan-PI3K inhibitors, alpelisib, and idelalisib also induced apoptosis in Kasumi-1 cells. In addition, we observed an increase in myeloid differentiation of Kasumi-1 cells with buparlisib or idelalisib. The splice isoform AML1-ETO9a is found in patients with t(8;21) AML, induces AML in mice when introduced into hematopoietic cells by retroviral transduction, and confers serial replating in methylcellulose (Yan et al, Nature Medicine 2006). To investigate the role of p110δ in leukemogenesis genetically, we harvested bone marrow cells from mice with a germline deletion of pik3cd, which encodes p110δ, and transduced them with a retrovirus expressing AML1-ETO9a. To study the ability of AML1-ETO9a leukemic cells to self-renew after p110δ deletion, we performed serial replating assays in methylcellulose.We found that, while AML1-ETO9a confers self-renewal activity to wild-type (WT) cells past round 8 of serial replating, p110δ-/- cells gradually lost self-renewal activity, and were unable to replate past round 4. Consistent with our genetic data, we found that the p110δ-selective inhibitor idelalisib or the pan-PI3K/mTOR inhibitor dactolisib (BEZ-235) can significantly reduce serial replating by AML1-ETO9a cells. To determine whether p110δ is also important for the initiation and progression of AML1-ETO AML in vivo, we transduced ckit-enriched bone marrow cells from WT or p110δ-/- mice with AML1-ETO9a-GFP retrovirus, and injected 200,000 GFP-sorted cells into lethally irradiated C57 Bl/6 recipient mice. During an interim analysis of this ongoing experiment, we have observed lower white cell counts in recipients of p110δ-/- cells compared with controls, suggesting a decrease in disease burden of AML1-ETO9a AML in the absence of p110δ.It has been reported that the thrombopoietin (TPO)/MPL/JAK2 signaling pathway is activated in t(8;21) AML, and is important in promoting proliferation and self-renewal of AML1-ETO leukemic cells (Pulikkan et al, Blood, 2012; Lo et al, Blood 2012). We hypothesized that p110δ may be required downstream of the TPO/MPL/JAK2 signaling pathway for leukemic self-renewal in t(8;21) AML. To determine whether TPO signaling depends upon p110δ in AML1-ETO9a cells, we treated WT and p110δ-/- cells transduced with AML1-ETO9a with TPO, and performed replating assays. We found that, although TPO treatment augmented the in vitro self-renewal of WT AML1-ETO9a cells to round 9 with increased colony numbers, p110δ-/- AML1-ETO9a cells treated with TPO failed to replate past round 5. These data suggest that p110δ is important for leukemic survival and self-renewal in AML1-ETO AML, in part by mediating signaling downstream of the TPO/MPL/JAK2 signaling axis. DisclosuresNo relevant conflicts of interest to declare.

Kinome Profiling Identifies Phospholipase C Gamma 1 (PLCγ1) As a Potential Target for t(8;21) AML

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. DisclosuresNo relevant conflicts of interest to declare.

ZGDHu-1 induces G₂/M phase arrest and apoptosis in Kasumi-1 cells.

The present study examined the effects of N,N′-di-(m-methylphenyi)-3, 6-dimethyl-1, 4-dihydro-1,2,4,5-tetrazine-1,4-dicarboamide (ZGDHu-1), a novel oxazine derivative, in Kasumi-1 cells. Following incubation with various concentrations of ZGDHu-1, fluorescence-activated cell sorting (FACS) was used in order to detect changes in mitochondrial membrane permeability in Kasumi-1 cells. Western blot analysis was performed in order to analyze the expression of nuclear factor-κB, inhibitor of κB and AML1/ETO. In addition FACS was used to analyze leukemia cell cycles and the expression levels of cyclin, cyclin-dependent kinases and cyclin-dependent kinase inhibitors in G2/M phase were determined using FACS and western blot analysis. The upregulation of reactive oxygen species production and mitochondrial membrane permeability was ascribed to apoptosis. The growth of Kasumi-1 cells was inhibited through the downregulation of nuclear factor-κB, degradation of AML1/ETO fusion protein and cell cycle arrest at the G2/M phase. This study documented that G2/M regulatory molecules, including cyclin B1, cell division control (cdc)2 and cdc25c were downregulated and checkpoint kinase 1 (CHK1), p53, p27, phospho-cdc25c, phospho-CHK1 and phospho-p53 were upregulated following treatment with ZGDHu-1. In the present study, pretreatment with CHIR-124, a selective CHK1 inhibitor, abrogated G2/M arrest via ZGDHu-1. These results demonstrated the anti-tumor activity of ZGDHu-1, which may therefore a potential target for further investigation and may be useful for the treatment of patients with t(8;21) acute myeloid leukemia.

Interference with RUNX1/ETO Leukemogenic Function by Cell-Penetrating Peptides Targeting the NHR2 Oligomerization Domain

The leukemia-associated fusion protein RUNX1/ETO is generated by the chromosomal translocation t(8;21) which appears in about 12% of all de novo acute myeloid leukemias (AMLs). Essential for the oncogenic potential of RUNX1/ETO is the oligomerization of the chimeric fusion protein through the nervy homology region 2 (NHR2) within ETO. In previous studies, we have shown that the intracellular expression of peptides containing the NHR2 domain inhibits RUNX1/ETO oligomerization, thereby preventing cell proliferation and inducing differentiation of RUNX1/ETO transformed cells. Here, we show that introduction of a recombinant TAT-NHR2 fusion polypeptide into the RUNX1/ETO growth-dependent myeloid cell line Kasumi-1 results in decreased cell proliferation and increased numbers of apoptotic cells. This effect was highly specific and mediated by binding the TAT-NHR2 peptide to ETO sequences, as TAT-polypeptides containing the oligomerization domain of BCR did not affect cell proliferation or apoptosis in Kasumi-1 cells. Thus, the selective interference with NHR2-mediated oligomerization by peptides represents a challenging but promising strategy for the inhibition of the leukemogenic potential of RUNX1/ETO in t(8;21)-positive leukemia.

Abstract 4351: p53 is a critical determinant of the antileukemic interactions between histone deacetylase inhibitors and chemotherapy drugs in pediatric acute myeloid leukemia cells

Abstract Acute myeloid leukemia (AML) accounts for one-fourth of acute leukemias in children, but it is responsible for more than half of the leukemia deaths in this patient population. Resistance to ara-C/anthracycline-based chemotherapy is a major cause of treatment failure in this disease. Among the newer agents that have been investigated in high-risk AML, clofarabine and valproic acid (VPA) are particularly notable. Clofarabine is a new FDA-approved drug for treating pediatric relapsed acute lymphoblastic leukemia and is currently being tested in pediatric AML. VPA is a FDA-licensed drug for treating both children and adults with epilepsy that has been shown to act as a histone deacetylase inhibitor (HDACI). We hypothesized that VPA and clofarabine might exhibit synergistic antileukemic activity in pediatric AML. In this study, we demonstrated highly synergistic antileukemic activities of combined clofarabine and VPA in 9 diagnostic blast samples from children with de novo AML. In contrast, both additive-to-synergistic [in Kasumi-1 which harbors t(8;21) and MV4-11 which harbors t(4;11)] and antagonistic (in THP-1 and CMS) results were obtained in 4 pediatric AML cell lines. AML cells which harbored t(8;21) were substantially more sensitive to VPA and showed greater responses to the drug combination than non-t(8;21) AML cells. Essentially the same results were obtained when clofarabine was combined with SAHA (a FDA-licensed pan-HDACI) in the pediatric AML cell lines. Co-treatment with clofarabine and VPA resulted in synergistic induction of apoptosis in Kasumi-1 cells, accompanied by synergistic activation of caspase-3 and cleavage of PARP. Our mechanistic studies revealed cooperative induction of DNA double-strand breaks (as reflected by induction of gH2AX), phosphorylation and acetylation of p53, and activation of Bax by clofarabine and VPA. Automated DNA sequencing revealed a p53 deficiency in THP-1 and CMS cell lines which may account for the antagonistic cytotoxic interactions between clofarabine and VPA, whereas the rest of the cells harbored either wild-type or mutant p53. Interestingly, treatments of Kasumi-1 cells with ara-C and VPA also resulted in cooperative activation of p53 and Bax, which may explain the differential responses of pediatric AML cells to combined ara-C and VPA observed in our previous study (Xie C et al. Clin Cancer Res 2010). Collectively, our results provide compelling evidence that p53 plays a key role in the synergistic cytotoxic effects of HDACIs combined with chemotherapy drugs in pediatric AML cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4351. doi:10.1158/1538-7445.AM2011-4351

Inhibitive effects of chimeric oncolytic adenovirus SG235 on leukemia cells in vitro

To investigate the inhibitive effects of chimeric oncolytic adenovirus SG235 on leukemia cells in vitro. The ability of SG235 to infect leukemia cells and the expression of CD46 on blasts from the patient with leukemia were detected by flow cytometry (FACS). The cytotoxicity of the virus was evaluated by MTT assay. Apoptosis induced by SG235 was detected with Annexin-V/PI staining and TUNEL assay followed by FACS analysis. The majority of leukemia cells from the patient with acute leukemia was CD46-positive. GFP-positive cells were 45.1%, 35.7%, 54.2%, 37.0%, 30.1%, %67.1, 17.2% and 33.1% in Mutz-1, Kasumi-1, K562, HL60, Molt- 4, RPMI8226, L428, and Jurkat cell lines treated with SG235-EGFP vector at MOI (multiplicity of infection) of 50 for 48 h.SG235 treatment resulted in marked growth inhibition and apoptosis of Kassumi-1 cells, and also significantly inhibited expression of p-Akt. The chimeric oncolytic adenovirus SG235 can infect leukemia cell effectively and results in the growth inhibition and apoptosis of Kasumi-1 cells in vitro.

Synergistic Antileukemic Interactions Between Valproic Acid and Cytarabine: A Potential Means to Improve the Treatment of Pediatric Acute Myeloid Leukemia.

Abstract 2084Poster Board II-61Acute myeloid leukemia (AML) accounts for one fourth of acute leukemias in children, but it is responsible for more than half of the leukemia deaths in this patient population. In contrast to the tremendous success in the treatment of acute lymphoblastic leukemia over the last three decades, resulting in a >80% cure rate, improvements in AML therapy have been limited. Resistance to cytarabine (ara-C), the most active drug in the treatment of AML, is a major cause of treatment failure in this disease. Therefore, new therapies for children with AML need to be developed to overcome drug resistance, decrease relapse rate, and reduce short- and long-term adverse effects of treatment. Histone deacetylase inhibitors (HDACIs) possess antitumor activity and are currently being tested in clinical trials for the treatment of a variety of different cancers. Valproic acid (VPA), an FDA-licensed drug for treating both children and adults with epilepsy, also acts as an HDACI and can induce apoptosis in leukemic cells but not normal cells. In this study, we hypothesized that VPA synergizes with ara-C in antileukemic activity by inducing apoptosis in AML cells. To model this concept and to provide the basis for future clinical studies, we examined the effects of VPA on sensitivities to ara-C in 8 AML cell lines derived from patients (4 were children) with different subtypes of AML and in AML blasts collected at the time of diagnosis from 10 children with de novo AML treated at Children's Hospital of Michigan. We demonstrated synergistic antileukemic interactions between ara-C and VPA in all of the AML cell lines and additive to synergistic antileukemic interactions between the two drugs in the patient samples by standard isobolograms and calculation of combination indexes. It is interesting to note that MV4-11 [which harbors t(4;11)] and Kasumi-1 [which harbors t(8;21)] cells were substantially more sensitive to VPA than the other AML cell lines. Analogous to the Kasumi-1 cells, diagnostic blasts from t(8;21) AML cases (n=4) were significantly more sensitive to VPA than blasts from non-t(8;21) AML cases (n=6) (mean VPA IC50 0.51 mM vs 1.95 mM, p=0.0095) and showed median 53.9-fold increased ara-C sensitivities when combined with VPA at concentrations of 0.5 mM or lower. By contrast, non-t(8;21) AML blasts only showed median 2.1-fold increased ara-C sensitivities when combined with 0.5 mM VPA (p=0.048). In a pilot experiment, treatment of SCID mice with K562 xenograft tumors with combined Palmo-ara-C and VPA resulted in a 31% T/C and a 0.8 gross log cell kill compared to treatments with Palmo-ara-C (67% T/C) or VPA alone (100% T/C), establishing unambiguous in vivo synergy. Real-time RT-PCR analyses revealed changes in transcript levels for hENT1 and cytidine deaminase in Kasumi-1 cells post VPA and ara-C treatment alone or in combination. However, these changes would antagonize ara-C sensitivity in Kasumi-1 cells, suggesting that the effects of VPA or ara-C alone or in combination on expression of genes related to ara-C transport and metabolism do not contribute to the observed synergistic effects in AML cells. Interestingly, ara-C and VPA co-treatment resulted in synergistic induction of apoptosis and S-phase arrest in Kasumi-1 cells determined by flow cytometry analysis with annexin V and PI staining. The synergy between ara-C and VPA in induction of apoptosis in Kasumi-1 cells was accompanied by synergistic activation of caspase-3, induction of both total and acetylated p53 (ac-p53), and release of the active form of Bax determined by caspase-3 assays, co-immunoprecipitation, and Western blotting. Collectively, these results suggest that VPA enhances ara-C sensitivity in Kasumi-1 cells most likely by modulating levels of total and ac-p53 proteins and then release of the active form of Bax to trigger apoptosis. Based on our laboratory results, VPA has been incorporated into a treatment arm for high risk AML patients enrolled in the St. Jude Children's Research Hospital (SJCRH) clinical trial AML08: “A Randomized Trial of Clofarabine Plus Cytarabine Versus Conventional Induction Therapy and of Natural Killer Cell Transplantation Versus Conventional Consolidation Therapy in Patients with Newly Diagnosed Acute Myeloid Leukemia”. The results of our study provide compelling evidence to support the use of VPA in combination with ara-C in clinical trials for treating different risk groups of pediatric AML. Disclosures:No relevant conflicts of interest to declare.

The Hsp90 inhibitor 17-allylamide-17-demethoxygeldanamycin induces apoptosis and differentiation of Kasumi-1 harboring the Asn822Lys KIT mutation and down-regulates KIT protein level

Heat shock protein 90 (Hsp90) serves as a chaperone for a number of cell signaling proteins, including many tyrosine and serine/threonine kinases, which are involved in proliferation and/or survival. The benzoquinone ansamycin geldanamycin has been shown to bind to Hsp90 and to specifically inhibit this chaperone's function, resulting in client protein destabilization. 17-Allylamino-17-demethoxygeldanamycin (17-AAG) is a chemical derivative of geldanamycin. KIT is the receptor for stem cell factor (SCF) and required for normal hematopoiesis. Mutations in c-Kit result in ligand-independent tyrosine kinase activity and uncontrolled cell proliferation. Kasumi-1 is t(8;21) acute myeloid leukemia (AML) cell line harboring mutated KIT with Asn822Lys substitution. Our present studies demonstrate that 17-AAG inhibits Kasumi-1 cells proliferation and exerts apoptosis- and differentiation-inducing effects in a dose- and time-dependent manner. The growth-inhibitory IC50 value for 17-AAG treatment is 0.62 μmol/L. Characteristic apoptotic features were confirmed by morphology, internucleosomal DNA fragmentation, and annexin V staining. 17-AAG also causes the G0/G1 block of Kasumi-1 cells. Significantly, 17-AAG-induced apoptosis of Kasumi-1 cells is associated with a decline in KIT protein level. Our findings strongly suggest that 17-AAG might be an effective therapeutic agent targeting AML cells harboring mutated KIT.

Induction of apoptosis in myeloid leukaemic cells by ribozymes targeted against AML1/MTG8.

The translocation (8;21)(q22;q22) is a karyotypic abnormality detected in acute myeloid leukaemia (AML) M2 and results in the formation of the chimeric fusion gene AML1/MTG8. We previously reported that two hammerhead ribozymes against AML1/MTG8 cleave this fusion transcript and also inhibit the proliferation of myeloid leukaemia cell line Kasumi-1 which possesses t(8;21)(q22;q22). In this study, we investigated the mechanisms of inhibition of proliferation in myeloid leukaemic cells with t(8;21)(q22;q22) by ribozymes. These ribozymes specifically inhibited the growth of Kasumi-1 cells, but did not affect the leukaemic cells without t(8;21)(q22;q22). We observed the morphological changes including chromatin condensation, fragmentation and the formation of apoptotic bodies in Kasumi-1 cells incubated with ribozymes for 7 days. In addition, DNA ladder formation was also detected after incubation with ribozymes which suggested the induction of apoptosis in Kasumi-1 cells by the AML1/MTG8 ribozymes. However, the ribozymes did not induce the expression of CD11b and CD14 antigens in Kasumi-1 cells. The above data suggest that these ribozymes therefore inhibit the growth of myeloid leukaemic cells with t(8;21)(q22;q22) by the induction of apoptosis, but not differentiation. We conclude therefore that the ribozymes targeted against AML1/MTG8 may have therapeutic potential for patients with AML carrying t(8;21)(q22;q22) while, in addition, the product of the chimeric gene is responsible for the pathogenesis of myeloid leukaemia. © 1999 Cancer Research Campaign