Cell Lines KG-1 Research Articles

Classifying Leukemias Based on Epigenetic Alterations.

Acute leukemia is caused by alterations of blood-forming stem cells leading to uncontrolled growth and diminished capacity to differentiate into mature functional blood elements. Beside genetic changes, epigenetic alterations are increasingly recognized as important events in the pathogenesis of leukemia. Cytosine methylation in CpG islands at gene transcription start regions can cause heritable gene silencing and have the same functional effects as inactivating mutations. Hundreds of genes may become epigenetically silenced in leukemia. While many of the methylated genes are not expressed in blood cells, silencing of genes critically important for control of stem cell self-renewal, proliferation, differentiation, and/or survival can contribute to the malignant phenotype. We used a genome-wide method to identify methylated genes by hybridizing a CpG island microarray with amplicons obtained by the methylated CpG island amplification technique (MCAM). We analyzed 10 leukemia cell lines with different cellular origin (myeloid cell lines KG1, KG1a, HEL, K562, and TF1; T lymphoid cell lines CEM and JTAg; and B lymphoid cell lines ALL1, BJAB, and Raji). On average, 266 genomic loci were found to be hypermethylated in these cell lines, ranging from 56 (KG1) to 483 loci (Raji), reinforcing the idea of extensive epigenome alteration in leukemia. Unsupervised hierarchical clustering showed distinct methylation pattern in the cell lines of lymphoid origin versus myeloid leukemia cell lines and a GM-CSF-dependent erythroleukemia cell line TF-1, justifying the use of methylation markers for uncovering of tumor-specific pathways of gene inactivation. There was a striking difference in the number of hypermethylated genes between two closely related myeloid leukemia cell lines: KG1 (56 methylated loci) and its undifferentiated variant KG1a (225 methylated loci). cDNA microarray analysis showed that deoxy-azacitidine treatment induced expression of genes differentially methylated in KG1a (DKKL1, GBX, HIVEP3, KCNAB1, KIAA1102, NAV2, NEIL1, and RAX) but not in KG1 cells where these genes were unmethylated. Finally, we used bisulfite PCR followed by pyrosequencing analysis to quantitatively measure DNA methylation of several genes detected by MCAM. Ongoing analyses of bone marrow samples from leukemia patients showed hypermethylation of the following genes: GDNF (in 4/22 [18%] AML and 7/20 [35%] ALL patients), HAND2 (in 5/22 [23%] AML and 7/20 [35%] ALL patients), HIVEP3 (in 9/22 [41%] AML and 6/20 [30%] ALL patients), MPDZ (in 2/6 [33%] AML and 15/20 [75%] ALL patients), and NEIL1 (in 2/20 [10%] AML and 1/12 [8%] ALL patients). Mapping of DNA methylation abnormalities may detect epigenetic markers important for leukemia classification and prognosis. Identification of pathways frequently silenced by DNA methylation may also suggest new targets for specific therapy.

Activity of TKI258 against primary cells and cell lines with FGFR1 fusion genes associated with the 8p11 myeloproliferative syndrome

AbstractThe 8p11 myeloproliferative syndrome (EMS) is an aggressive, atypical stem cell myeloproliferative disorder associated with chromosome translocations that disrupt and constitutively activate FGFR1 by fusion to diverse partner genes. To explore the possibility of targeted therapy for EMS, we have investigated the use of TKI258, a multitargeted receptor tyrosine kinase inhibitor with activity against FGFR, VEGFR, PDGFR, FLT3, and KIT that is currently being assessed for the treatment of a variety of malignancies in phase 1 clinical studies. The viability of Ba/F3 cells transformed to IL3 independence by ZNF198-FGFR1 or BCR-FGFR1 was specifically inhibited by TKI258 with IC50 values of 150 nM and 90 nM, respectively. Inhibition was accompanied by dose-dependent inhibition of phosphorylation of each fusion gene, ERK, and STAT5. TKI258 also specifically inhibited proliferation and survival of the FGFR1OP2-FGFR1–positive KG1 and KG1A cell lines, resulting in increased levels of apoptosis. Primary cells from EMS patients showed significant, dose-dependent responses in liquid culture and in methylcellulose colony assays compared with controls. This work provides evidence that targeted therapy may be beneficial for patients with EMS.

Phenotypic and functional differentiation of KG-1 into dendritic-like cells

The cell line KG-1 has been used as an in vitro model for human dendritic cell (DC) differentiation. We have investigated the response of KG-1 cells to stimulation with a number of factors known to induce differentiation and/or maturation of DCs in vitro. KG-1 cells showed no differentiation in response to LPS, CpG oligodeoxynucleotide or CD40 ligation. Culture in the presence of TNF- α induced some differentiation, but only treatment with PMA and ionomycin (with or without prior culture in GM-CSF and IL-4) induced morphological and phenotypic changes consistent with DC-like maturation, and even these maximally differentiated KG-1 cells showed lower levels of surface marker expression, macromolecular endocytosis, and ability to stimulate in allogeneic MLR compared with in vitro monocyte-derived DCs. Our data show that KG-1 cells differentiate in vitro into cells with DC-like functional characteristics under the influence of strong inducers of cellular activation, but lack the potency of mature DCs in key aspects of professional antigen presenting cells.

Major Histocompatibility Complex Class I-ERp57-Tapasin Interactions within the Peptide-loading Complex

The endoplasmic reticulum-located multimolecular peptide-loading complex functions to load optimal peptides onto major histocompatibility complex (MHC) class I molecules for presentation to CD8(+) T lymphocytes. Two oxidoreductases, ERp57 and protein-disulfide isomerase, are known to be components of the peptide-loading complex. Within the peptide-loading complex ERp57 is normally found disulfide-linked to tapasin, through one of its two thioredoxin-like redox motifs. We describe here a novel trimeric complex that disulfide links together MHC class I heavy chain, ERp57 and tapasin, and that is found in association with the transporter associated with antigen processing peptide transporter. The trimeric complex normally represents a small subset of the total ERp57-tapasin pool but can be significantly increased by altering intracellular oxidizing conditions. Direct mutation of a conserved structural cysteine residue implicates an interaction between ERp57 and the MHC class I peptide-binding groove. Taken together, our studies demonstrate for the first time that ERp57 directly interacts with MHC class I molecules within the peptide-loading complex and suggest that ERp57 and protein-disulfide isomerase act in concert to regulate the redox status of MHC class I during antigen presentation.

Effect of hepatocyte growth factor (HGF) on the level of Survivin & XIAP expression in several human cancer cell lines, after treating with DNA damaging agent

Hepatocyte growth factor (HGF) has opposite biological activities in regulating apoptosis, also underlying molecular mechanisms are not clearly defined. We investigated HGF ability to inhibit cell death, which was induced by Doxorubicin, a DNA damaging agent. Also Survivin and XIAP mRNA levels were compared in HGF treated and non-treated cells. Cell proliferation and death were assessed using MTT assay and dye exclusion tests. Quantitative real-time PCR was used to evaluate Survivin and XIAP expression levels after treatment with HGF. ELISA was performed to quantify HGF secretion in the selected cancer cell lines media. HGF appeared to have inhibitory effect on Doxorubicin induced cell death in all of the studied cell lines. It had minimal effect on XAIP and Survivin expression levels in MRC-5, MOLT-4 and AGS cell lines; except for XIAP expression level in AGS cell line, which was increased substantially after treatment. Surprisingly, in KG-1 cell line, XIAP and Survivin expression levels were significantly reduced after HGF treatment. Although several members of IAP gene family are reported to play role in HGF mediated cytoprotective pathway, we showed that XIAP and Survivin do not seem to be involved.

Discovery of epigenetically silenced genes in acute myeloid leukemias

The demethylating 5-aza-2'deoxycytidine (DAC) and the histone deacetylase inhibitor (HDACi) suberoyl anilide bishydroxamide (SAHA) possess potent antitumorigenic properties in myeloid disorders. However, the transcriptome alterations mediated by these drugs are poorly understood. We analyzed the transcriptional effects of DAC and SAHA in the AML cell line KG-1. Microarray analyses revealed 76 genes expressed in normal CD34+ cells, absent in KG-1 cells but whose expression was induced after drug treatment. A total of 39 of these genes harbored CpG islands in their promoters. We examined the expression level of these genes in 120 AML patient samples representing diverse karyotpyes. Gas2l1, tfIIs, ehd3, enolase 2, mx1, dral, astml and pxdn were diminished across all AML karyotypes examined. Ehd3 was methylated in 63% of AML patients examined. This methylation was lost upon complete remission, and not observed in normal CD34+ cells. CD34+ cells expressed ehd3 at approximately 10-fold higher levels than AML samples. Another highlighted gene, alpha-catenin, is located at q31 of chromosome 5. Analyses of 29 5q- AML/myelodysplastic syndrome (MDS) samples revealed marked decreases in expression of alpha-catenin, compared to non-5q- MDS samples (6.6+/-9-fold). However, no methylation was detected, suggesting indirect effects of these drugs on the expression of alpha-catenin.

Gene Expression Analysis of Hematopoietic Progenitor Cells Identifies Dlg7as a Potential Stem Cell Gene

Inducible hematopoietic stem/progenitor cell lines represent a model for studying genes involved in self-renewal and differentiation. Here, gene expression was studied in the inducible human CD34+ acute myelogenous leukemia cell line KG1 using oligonucleotide arrays and suppression subtractive cloning. Using this approach, we identified Dlg7, the homolog of the Drosophila Dlg1 tumor suppressor gene, as downregulated at the early stages of KG1 differentiation. Similarly, Dlg7 was expressed in normal purified umbilical cord blood CD34+CD38- progenitors but not in the more committed CD34+CD38+ population. Dlg7 expression was not detected in differentiated cells obtained from hematopoietic colonies, nor was expression detected in purified T-cells, B-cells, and monocytes. When analyzed in different types of stem cells, Dlg7 expression was detected in purified human bone marrow-derived CD133+ progenitor cells, human mesenchymal stem cells, and mouse embryonic stem (ES) cells. Overexpression of Dlg7 in mouse ES cells increased their growth rate and reduced the number of EBs emerging upon differentiation. In addition, the EBs were significantly smaller, indicating an inhibition in differentiation. This inhibition was further supported by higher expression of Bmp4, Oct4, Rex1, and Nanog in EBs overexpressing Dlg7 and lower expression of Brachyury. Finally, the Dlg7 protein was detected in liver and colon carcinoma tumors but not in normal adjacent tissues, suggesting a role for the gene in carcinogenesis. In conclusion, our results suggest that Dlg7 has a role in stem cell survival, in maintaining stem cell properties, and in carcinogenesis. Disclosure of potential conflicts of interest is found at the end of this article.

Interleukin-18 directly activates T-bet expression and function via p38 mitogen-activated protein kinase and nuclear factor-κB in acute myeloid leukemia–derived predendritic KG-1 cells

The leukemic cell line KG-1 was isolated from a patient with acute myeloid leukemia and is regarded a cellular model of human dendritic cell progenitors. The T helper type 1 cytokine interleukin (IL)-18 has been shown to induce the maturation of these cells towards a dendritic phenotype and, moreover, is able to mediate IFNgamma production in this model. Because T-box expressed in T cells (T-bet) is considered to be of paramount importance for dendritic cell function, the effects of IL-18 on this transcription factor have been investigated in the current study. Here, we show that activation of KG-1 cells by IL-18 induces T-bet mRNA and protein within 4 to 6 h of incubation. This hitherto unrecognized function of IL-18 was suppressed by the inhibition of p38 mitogen-activated protein kinase activity and nuclear factor-kappaB function. Blockage of translation by cycloheximide, usage of neutralizing antibodies, and the inability of IFNgamma to mediate significant p38 mitogen-activated protein kinase activation in KG-1 cells clearly revealed that activation of T-bet was not via autocrine IFNgamma. T-bet function was evaluated by short interfering RNA technology. Notably, specific suppression of T-bet induction impaired secretion of IFNgamma by KG-1 cells under the influence of IL-18. Therapeutic application of IL-18 has the potential to profoundly affect the biology of acute myeloid leukemia predendritic cells such as KG-1 cells. Under these conditions, activation of T-bet may play a key role in processes that have the potential to correct the T helper type 1 deficiency associated with leukemia-mediated immunosuppression.

Expression and mutation analysis of genes that encode the Myc antagonists Mad1, Mxi1 and Rox in acute leukaemia

The Myc antagonists Mad1, Mxi1 and Rox proteins share two highly conserved domains, Sin3-interacting domain (SID) and basic helix-loop-helix leucine zipper domain (bHLHzip), which are essential for these proteins to function during molecular switching from proliferation to differentiation. In an attempt to identify mutations in Mad1, Mxi1 and Rox genes in human haematological malignancies, we screened 10 haematopoietic cell lines, bone marrow mononuclear cells (BMMNC) from 26 patients with haematological malignancies and peripheral blood mononuclear cells (PBMNC) from 30 healthy volunteers, using reverse transcription-polymerase chain reaction, single strand conformation polymorphism analysis and sequencing. Mad1, Mxi1 and Rox genes were expressed in all samples. Four polymorphisms were found in cell lines BMMNC and PBMNC: two in Mad1, one in Mxi1 and one in Rox. Nine missense mutations were detected: two in Mad1 in patients, four in Mxi1 (three in patients and one in KG-1 cell line), and three in Rox in patients. No mutations were detected in PBMNC from healthy volunteers. Among six patients with acute lymphoblastic leukaemia, two had Mxi1 mutations and another two had Rox mutations. These mutations were associated with poorer clinical outcomes. This is the first report to show that Mad1, Mxi1 and Rox genes were expressed and displayed mutations in haematological malignancies.

Phosphotyrosine profiling identifies the KG-1 cell line as a model for the study of FGFR1 fusions in acute myeloid leukemia

AbstractThe 8p11 myeloproliferative syndrome (EMS) is associated with translocations that disrupt the FGFR1 gene. To date, 8 fusion partners of FGFR1 have been identified. However, no primary leukemia cell lines were identified that contain any of these fusions. Here, we screened more than 40 acute myeloid leukemia cell lines for constitutive phosphorylation of STAT5 and applied an immunoaffinity profiling strategy to identify tyrosine-phosphorylated proteins in the KG-1 cell line. Mass spectrometry analysis of KG-1 cells revealed aberrant tyrosine phosphorylation of FGFR1. Subsequent analysis led to the identification of a fusion of the FGFR1OP2 gene to the FGFR1 gene. Small interfering RNA (siRNA) against FGFR1 specifically inhibited the growth and induced apoptosis of KG-1 cells. Thus, the KG-1 cell line provides an in vitro model for the study of FGFR1 fusions associated with leukemia and for the analysis of small molecule inhibitors against FGFR1 fusions.

Inhibition of Bcl-2 Signaling by Small Molecule BH3 Inhhibitor GX15-070 as a Novel Therapeutic Strategy in AML.

GX15-070 is a novel cycloprodigiosin derived small molecule BH3 inhibitor that binds with moderate affinity to all antiapoptotic Bcl-2 family members, including Mcl-1, and is currently undergoing Phase I clinical trials in leukemias. In this study, we investigated the activity of GX15-070 in acute myeloid leukemia (AML) cell lines and primary AML samples. GX15-070 inhibited cell growth of HL-60, U937, OCI-AML3 and KG-1 cell lines at IC50's of 0.1, 0.5, 0.5 and 2.5μM, respectively, at 72 hours. Neither overexpression of Bcl-2 or Bcl-XL nor loss of expression of Bax conferred resistance to GX15-070. GX15-070 inhibited Bim/Bcl-2 heterodimerization and induced association of activated Bak with Bax in OCI-AML3 cells, as demonstrated by co-immunoprecipitation studies using CHAPS buffer. This was associated with cytosolic release of cytochrome c followed by an increase in annexin positivity, caspase activation and a decrease in mitochondrial inner membrane potential. Notably, GX15-070 induced cytochrome c release from isolated mitochondria of leukemic cells. GX15-070 synergized with both AraC and the novel BH3 mimetic ABT-737 to induce apoptosis in OCI-AML3 cells, a notoriously chemoresistant cell line (GX15-070 and ABT-737 average CI value 0.3; GX15-070 and AraC average CI value 0.36). In 6/7 primary AML samples, GX15-070 induced apoptosis in CD34+ progenitor cells at an average IC50 of 3.6±1.2μM at 24 hours. GX15-070 potently inhibited clonogenic ability of AML blasts at sub-micromolar doses (58.5±10.6% CFU-Blast at 0.1μM and 38.1±10.5% at 0.25μM, n=7). In summary, BH3 inhibitor GX15-070 induces apoptosis in AML cells via inhibition of association of pro-survival Bcl-2 family proteins and BH3-only proteins, followed by Bax/Bak activation and initiation of the intrinsic apoptotic pathway. Hence, GX15-070 alone or in combination with chemotherapeutic agents may have utility in AML therapy.

Lyn Kinase Is Constitutively Activated, Controls the mTOR/p70S6K/4E-BP1 Pathway and Regulates Cell Proliferation in Acute Myeloid Leukemia.

We have recently demonstrated that the mTOR/4E-BP1/p70S6K pathway is constitutively activated in AML cells but not in their normal counterpart. Rapamycin inhibits AML cell proliferation in 60% of cases and induces transient blood and marrow blast clearance in refractory AML patients (Blood 2005; 105:2527–34). However, exact mechanisms of activation of this pathway in AML are poorly understood. Since receptor or non-receptor tyrosine kinases (TK) play a crucial role in leukemogenesis and may activate downstream effectors such as mTOR/4E-BP1/p70S6K, we have studied the basal profile of protein tyrosine phosphorylations in a series of fresh samples and the effect of several TK inhibitors on mTOR targets in order to identify upstream regulators of this pathway.PP2 (Src Familly Kinases, SFK inhibitor), herbimycin A (pan TK) but not SU6656 (pp60 c-src), AG1296 (c-Kit, PDGFR, FLT3) and AG490 (JAK2) inhibited the phosphorylation of p70S6K and 4E-BP1 in KG1 cell line. PP2 consistently inhibited the phosphorylation of mTOR targets in 30 fresh AML samples. Using immunostaining with anti-phosphotyrosine antibody, we detected a high level of tyrosine phosphorylations in all AML samples (in contrast to normal CD34+cells) and consistently found a signal at 55–60 kD which may correspond to SFK. Moreover, PP2 dramatically reduced the global level of tyrosine phosphorylations suggesting that SFK play a role on TKs activation cascade and could act upstream mTOR.Lyn, Hck and Fgr were expressed in 100%,78% and 67% of cases while Lck and Fyn were weakly or not expressed in AML cells. All samples showed phosphorylation on Y416 residue demonstrating the constitutive activation of SFK. We also used flow cytometry to detect SFK phosphorylation in the more immature compartment of the leukemic clone. In 4/5 cases, the CD34+CD38−CD123+ cell sub population displayed a high level of phosphorylation on Y416 that was inhibited by PP2. In KG1, PP2 inhibited cell growth in a time and dose dependent manner. This effect was mainly due to a cell cycle arrest in G1 and to a lesser extent, apoptosis induction. In clonogenic assays, PP2 inhibited the capacity of fresh AML cells to generate AML-CFU and induced apoptosis in short term culture (n=6).Because the two Lyn kinase isoforms were expressed in all cases and corresponded to the two bands detected by p-SrcY416 antibody, we thought to determine if this this kinase was the major component of SFK in AML. Lyn was highly phosphorylated on Y416 after immunoprecipitation. Specific down regulation by si-RNA to Lyn induced a significant decrease of p70S6K and 4E-BP1 phosphorylation and inhibited cell proliferation in KG1 and U937. Using confocal microscopy, Lyn was mainly localized all over the plasma membrane and colocalized with anti-p-SrcY416 whereas its distribution in normal CD34+ cells was restricted to several distinct points across the membrane.These data demonstrate that SFK and particularly Lyn kinase, are aberrantly deregulated in AML cells, control cell proliferation at least in part through mTOR pathway and represent a new target for the treatment of this disease.

Human CD34+ Myeloid Leukemic Progenitor Cells Are Susceptible to Lysis by Minor Histocompatibility Antigen LRH-1-Specific Cytotoxic T Lymphocytes.

Allogeneic stem cell transplantation (SCT) is a specialized form of immunotherapy for treating patients with hematological malignancies. The curative potential is attributed to the graft-versus-tumor (GVT) response during which donor-derived cytotoxic T lymphocytes (CTL) eliminate malignant cells of the recipient. Minor histocompatibility antigens (MiHA) are the major targets of the GVT response, and expansion of MiHA-specific CTL has been shown to coincide with tumor remission following SCT. Recently, we identified a novel hematopoietic cell-restricted MiHA, designated LRH-1, which is presented by HLA-B7 and encoded by the P2X5 purinergic receptor gene (J. Clin. Invest. 2005: 115:3506–3516). Interestingly, tetramer analysis showed a direct association between in vivo expansion of LRH-1-specific CD8+ T cells and the disappearance of Bcr-Abl positive tumor cells in the CML patient from whom LRH-1-specific CTL was originally isolated. In addition, we detected in vivo expansion of LRH-1-specific CTL in an AML patient who was in clinical remission without GVHD. Furthermore, we demonstrated that P2X5 mRNA is significantly expressed in leukemic CD34+ progenitor cells from most CML as well as AML patients. These findings indicate a role for LRH-1 in inducing GVT immunity against myeloid leukemic progenitor cells. Here, we investigated the ex vivo responsiveness of myeloid leukemic CD34+ progenitor cells to LRH-1-specific CTL. First, we addressed this question using the CD34+ KG1 cell line which is positive for LRH-1. By using a CFSE-based survival assay we demonstrated that KG1 cells stably transfected with HLA-B7 could be efficiently lysed by LRH-1-specific CTL. Next, we determined responsiveness of purified CD34+ progenitor cells from HLA-B7+ CML patients to LRH-1 CTL-mediated killing. In the CFSE-based survival assay as well as a hematopoietic progenitor cell inhibition assay we showed that LRH-1-specific CTL efficiently recognize and kill CD34+ progenitor cells from LRH-1+ CML patients. In contrast, LRH-1-specific CTL did not inhibit the proliferation of CD34+ progenitor cells from LRH-1- CML patients. These findings illustrate that the P2X5-encoded LRH-1 antigen is an attractive target for adequate eradication of myeloid leukemic progenitor cells after allogeneic SCT.

A Role for DNA Double Strand Breaks in the Sensitivity of AML Cells to Clofarabine: Enhanced In Vitro Sensitivity in Primary AML Cells with FLT3 Mutations.

Clofarabine is a purine nucleoside analogue which has been incorporated into several therapeutic trial protocols for the treatment of leukaemias including acute myeloid leukaemia (AML). The aims of the study are to ascertain mechanisms of clofarabine action in AML using cell lines and presentation samples from patients. We measured apoptosis by TdT assay, cytochrome C release and flow cytometric assays of the mitochondrial membrane potential probe DiOC6. To study the effects of clofarabine on DNA synthesis and DNA double strand breaks, we used bromodeoxyuridine (BrdU) and H2AX assays respectively. Equitoxic doses were established that caused approximately 20% cell death in the AML cell lines HL-60 (0.3 μM), KG1 (1 μM) and MV4.11 (1 μM) after 6 hours of continuous exposure. At these doses, clofarabine induced apoptotic DNA nicks, as measured by the TdT assay, and Cytochrome C release in all three cell lines. However, clofarabine-induced mitochondrial hyperpolarisation and depolarisation were found to be cell-type specific, occurring in HL-60 but not MV4.11 or KG1 cells, i.e. mitochondrial membrane depolarisation is not an essential part of the mechanism of clofarabine-induced apoptosis. Following clofarabine treatment, DNA synthesis was reduced by 91% within 1 hour in KG1 cells and by 80% in HL-60, but by only 9% in MV4.11. Out of the three cell lines, MV4.11 cells, which have a FLT3 internal tandem duplication (ITD), were the only ones to completely repair DNA double strand breaks following clofarabine removal. In 12 samples from patients with AML, the proportion of cells which incorporated BrdU in a 45 minute assay - a measure of the rate of cell cycling - differed considerably between samples, from 0.8% to 23%, median 13%. Cell death induced by clofarabine was correlated with the cycling rate of untreated cells (P=0.016). All 12 samples showed inhibition of DNA synthesis within 60 minutes of clofarabine treatment (range 20%–92% inhibition, median 54%). Mitochondrial membrane hyperpolarisation and depolarisation were not observed in patient cells. However, DNA double strand breaks were induced by clofarabine in patient cells. Paradoxically, i.e. in contrast to the results seen with the MV4.11 cell line, toxicity was greatest in samples with a FLT3 mutation (P=0.007). In conclusion, the FLT3 mutant MV4.11 cell line effectively repairs clofarabine-induced double strand breaks. Cell death in patient cells cultured with clofarabine is correlated with the presence of a FLT3 mutation. As we have previously established that AML samples with FLT3 mutations have upregulated DNA repair activity, this paradox might be explained by cycles of attempted DNA repair frustrated by renewed clofarabine incoporation into DNA, thus increasing the toxicity of the drug.

Molecular and Functional Effects of the Novel MEK Inhibitor PD0325901 in Preclinical Models of Human Leukemias.

The Raf/MEK/ERK signaling module plays a pivotal role in the regulation of cell proliferation, survival, and differentiation. Our group, among others, has recently demonstrated that this pathway is frequently dysregulated in hematological malignancies and may constitute an attractive therapeutic target, particularly in AML. Here we investigated the effects of PD0325901, a novel MEK inhibitor, on phospho-protein expression, gene expression profiles, cell proliferation, and apoptosis in cell line models of AML, ALL, multiple myeloma (MM), ex vivo-cultured primary AML blasts, and oncogene-transformed hematopoietic cells. AML cell lines (OCI-AML2, OCI-AML3, HL-60) were strikingly sensitive to PD0325901 (IC50: 5–19 nM), NB4 (APL) and U266 (MM) showed intermediate sensitivity (IC50: 822 and 724 nM), while all the lymphoid cell lines tested and the myeloid cell lines U937 and KG1 were resistant (IC50 > 1000 nM). Cell growth inhibition was due to inhibition of cell cycle progression and induction of apoptosis. A statistically significant reduction in the proportion of S-phase cells (p=0.01) and increase in the percentage of apoptotic cells (p=0.019) was also observed in 18 primary AML samples in response to 100 nM PD0325901. Analysis of the correlation between sensitivity/resistance to PD0325901 and Ras/Raf mutation status is currently ongoing. PD0325901 effects were also examined in a panel of IL-3-dependent murine myeloid FDC-P1 cell lines transformed to grow in response to 11 different oncogenes in the absence of IL-3. Fms-, Ras-, Raf-1-, B-Raf-, MEK1-, IGF-1R-, and STAT5a-transformed FDC-P1 cells were very sensitive to PD0325901 (IC50: ~ 1 nM), while A-Raf-, BCR-ABL-, EGFR- or Src-transformed cells were 10 to 100 fold less sensitive (IC50: 10 to 100 nM); the parental, IL-3 dependent FDC-P1 cell line had an IC50 > 1000 nM. Analysis of the phosphorylation levels of 18 different target proteins after treatment with 10 nM PD0325901 showed a 5- to 8-fold reduction in ERK-1/2, observed only in sensitive cell lines, and a 2-fold reduction in JNK and STAT3 phosphorylation. PD0325901 (10 nM) treatment also profoundly altered the gene expression profile of the sensitive cell line OCI-AML3: 96 genes were modulated after 24 h (37 up- and 59 down-regulated), most of which involved in cell cycle regulation. Changes in cyclin D1 and D3, cyclin E, and cdc 25A were also validated at the protein level. Overall, PD0325901 shows potent growth-inhibitory and pro-apoptotic activity, indicating that MEK may be an appropriate therapeutic target in an array of different hematological malignancies. Further preclinical/clinical development of this compound is warranted, particularly in myeloid leukemias.

Targeting of Heat Shock Protein 32 (Hsp32) in Neoplastic Cells by Styrene Maleic Acid Zinc Protoporphyrin (SMA-ZnPP) Is Associated with Reduced Growth and Induction of Apoptosis.

Heat shock protein 32 (Hsp32), also known as heme oxygenase-1 (HO-1), is a stress-related survival factor that has recently been implicated in enhanced survival of neoplastic cells. We here show that Hsp32/HO-1 is expressed abundantly in primary neoplastic cells in various solid tumors and hematopoietic neoplasms such as acute myeloid leukemia (AML) or chronic myeloid leukemia (CML), and in respective cell lines including the AML cell lines HL60, KG1, KG1a, and U937, CML cell lines K562 and KU812, eosinophilic leukemia cell line EOL-1, mast cell leukemia cell line HMC-1, myeloma cell lines RPMI8226 and U266, breast cancer cell line MDA MB 231, lung cancer cell line A549, pancreatic carcinoma cell line BxPC-3, colon carcinoma cell lines COLO201, COLO205, COLO320DM, and DLD-1, and the ovarian carcinoma cell line OVCAR-3. Expression of Hsp32 mRNA was demonstrable by RT-PCR and Northern blotting, and expression of the Hsp32 protein by Western blotting and immunocytochemistry. The CML-specific oncoprotein BCR/ABL and the transforming oncoprotein KIT D816V that is expressed in neoplastic mast cells, were found to promote expression of Hsp32 in Ba/F3 cells. In order to examine the functional role of Hsp32 in neoplastic cells, a specific siRNA was employed. Expression of Hsp32 siRNA resulted in reduced viability and induction of apoptosis in all cell lines tested. To further explore the value of Hsp32 as a target in neoplastic cells, a novel specific Hsp32-targeting compound, styrene maleic acid copolymer zinc protoporphyrin micelles (SMA-ZnPP) was applied. Exposure to SMA-ZnPP resulted in a significant decrease in proliferation determined by 3H-thymidine uptake, in all cell lines as well as in all primary neoplastic cells tested (AML, n=5; CML, n=5; mastocytosis, n=3; breast cancer, n=2; lung cancer, n=1). As assessed by AnnexinV-staining, Tunel assay and electron microscopy, the growth-inhibitory effects of SMA-ZnPP were found to be associated with induction of apoptosis. In each cell type, the effect of SMA-ZnPP on growth was dose-dependent and found to occur at pharmacologic concentrations (IC50 1–30 μM). Moreover, SMA-ZnPP was found to synergize with various anti-neoplastic drugs (tumor cell lines: cisplatin, leukemias: cytarabine, myeloma: bortezomib) in producing growth inhibition. In summary, these data show that Hsp32/HO-1 is an important survival factor and novel interesting target in various hematopoietic and non-hematopoietic neoplasms. Based on these data, it seems desirable to explore the value of the Hsp32-targeting drug SMA-ZnPP in clinical trials in patients with leukemias and solid tumors.

Inhibition of Cellular Aminopeptidases as Novel Therapy for AML.

CHR 2797 is one of a new class of enzyme inhibitors with a pleiotropic effect against a number of human cancer cells. It is thought to inhibit the M1 family of metalloenzymes that include aminopeptidases, and is under investigation for the treatment of acute myeloid leukemia. Aminopeptidases catalyse the hydrolysis of the terminal amino acids from short chain polypeptides and they are involved in the continuous cycle of protein formation and degradation in cells. As malignant cells are thought to be more highly dependant on this protein cycling, interrupting this pathway is therefore a potential therapeutic target for novel agents. The effects of the aminopeptidase inhibitor CHR 2797 were investigated in AML cells in-vitro. Leukemic cells and cell lines were treated with CHR 2797 at a range of 0.0002 – 20μM and IC50 values were calculated from the WST-1 proliferation experiments. The AML cell lines HL60, KG1, K562 and U937 had an average IC50 of 1μM with a range between 0.01 and 10μM. Primary diagnostic AML samples (n=40) were analysed and an IC50 range of between 0.01 and >40μM were detected, with a median of 0.8μM. The effects of CHR 2797 were also analysed on normal bone marrow samples (n=10). The IC50 range was between 6.2 and >40μM with a median of 15μM, demonstrating a potential therapeutic window between the treatment of the leukemic cells and toxicity to the normal samples. The level of synergy or antagonism with conventional therapeutic agents was calculated using a combination index. Synergy was demonstrated in 70% of cell samples in combination with ARA-C, and 80% with Velcade. Synergy was also shown in 60% of cells samples with ATRA, even in non-promyelocytic leukemia types. Annexin V and cell cycle analysis confirmed apoptosis after treatment with CHR 2797 in many cases. A degree of differentiation of acute promyelocytic cells to mature myeloid cells was also stimulated with the treatment. The effects of CHR 2797 on cellular aminopeptidases were also measured. CD13 is a cell surface protein which is expressed selectively on myeloid cells and is also classified as an aminopeptidase N. Its activity can be measured by the conversion of the substrate ala-MCA to the protein MCA that can be detected on a fluorometric plate reader. CHR 2797 was demonstrated to reduce CD13 activity in a time and dose responsive manner. The reduction in activity was demonstrated immediately following addition of the drug, and a persistent effect was shown over four days of cell culture. A reduction in CD13 activity was also shown with a concentration of CHR 2797 of under 0.5μM; and with a 10μM dose the activity in many AML samples was reduced by >90%. New treatments are needed for acute myeloid leukemia to improve survival and reduce the toxicity of conventional therapy. This study demonstrates that CHR 2797 might be an effective molecular therapy for AML, either alone or in combination with other chemotherapeutic agents.

Sustained Expansion of Human Natural Killer Cells for Leukemia Therapy.

Natural killer (NK) cells are a promising tool for cell therapy of hematologic malignancies. They have potential for enhancing graft-versus-leukemia responses in recipients of hematopoietic stem cell transplant, and can also be used in a non-transplant setting, where haploidentical donor NK cells have been shown to expand in vivo. NK cells represent a small subset of peripheral blood cells. Hence, it can be problematic to obtain them in quantities sufficient to exert significant anti-leukemic activity in patients. We sought to identify culture conditions that would stimulate vigorous, sustained and specific expansion of CD56+ CD3− NK cells. NK cell proliferation was stimulated by contact with the K562 leukemia cell line transfected with two NK stimulatory molecules: membrane-bound interleukin 15 and 4-1BB ligand. Exposure of peripheral blood cells from 23 donors to irradiated K562-mb15-41BBL cells in the presence of 10 IU/mL interleukin-2 resulted in a median expansion of CD56+ CD3− cells of 22-fold (range, 9- to 87-fold) after only 7 days of culture; expansion of CD3+ T cells was negligible. After 14 days of culture, K562-mb15-41BBL cells were completely lysed by the NK cells and no further expansion occurred. However, further NK cell expansion could be achieved by addition of fresh K562-mb15-41BBL cells to the cultures. Using this method, NK cell expansions ranged from 2,000- to 98,000-fold (n = 4) after 65 days of culture. We noted that NK cells eventually became unresponsive to stimulation and underwent senescence after 2–5 months of culture. To determine whether NK cell senescence could be overcome by enforced expression of human telomerase reverse transcriptase (hTERT), we stimulated NK cells for 1 week with K562-mb15-41BBL cells and then transfected them using an MSCV retroviral vector and the hTERT gene (gift of Dr. J. Dome, St. Jude). hTERT expression and telomerase activity was demonstrated by reverse transcriptase-polymerase chain reaction and telomerase repeat amplification protocol assay. The cultures were then stimulated with periodic pulses of K562-mb15-41BBL cells. In 2 donors, enforced expression of hTERT overcame senescence: NK cells transfected with an empty vector died after 85 and 170 days of culture, whereas hTERT-NK cells continue to grow after more than 350 days of culture, while retaining a normal karyotype. hTERT-NK cells maintained their cytotoxicity against the NK-sensitive leukemic cell lines K562, KG1, U937, HL60 and Jurkat. They could be also be genetically modified to express anti-CD19 chimeric signaling receptors, thus becoming cytotoxic against NK-resistant CD19+ B-lineage acute lymphoblastic leukemia cells. Cytotoxicity against CD19+ targets was similar to that of NK cells transfected with the signaling receptor after only one week of culture. In conclusion, coculture of human peripheral blood mononuclear cells with pulses of irradiated K562-mb15-41BBL cells allows the generation of a large numbers of NK cells which have powerful anti-leukemic capacity and can be redirected to lyse NK-resistant target cells. Although senescence eventually ensues, this can be overcome by hTERT expression. The culture system described here has now been adapted to large scale expansion for clinical use.

AS1411, a Novel DNA Aptamer as a Potential Treatment of Acute Myelogenous Leukaemia (AML).

AS1411 (formerly AGRO100) is the first nucleic acid aptamer to enter oncology trials and is currently in a Phase I study in renal and lung patients. AS1411 is a 26mer unmodified phosphodiester-based guanosine rich oligonucleotide which, as a dimer, forms a serum-resistant quartet structure. It is postulated to exert anti-cancer activity by binding to cell surface nucleolin resulting in S-phase cell cycle arrest. We have previously shown that AS1411 inhibits growth of a wide range of solid tumours, including lung, renal, prostate and colorectal with EC50s of 2 to 8 μM. In this series of studies, we investigated the potential of AS1411 in the treatment of acute myelogenous leukaemia (AML). Expression of cell-surface and intracellular expression of nucleolin was assessed by indirect immunocytochemical staining. Four AML cell lines (MV4-11, KG1, HL60 and K562) and two solid tumour cell lines (A549 - lung; DU145 - prostate) were used. Permeabilised or non-permeabilised cells were either stained in situ on chamber glass slides or as cytospin preparations. Three anti-nucleolin monoclonal antibodies and the Vector immunoperoxidase system were used. Cells were qualitatively assessed for extent and intensity of staining. In the KG1, HL60 and MV4-11 cell lines, intense, homogeneous staining was seen in 100% of non-permeabilised cells, indicating high levels of surface-expressed nucleolin; in K562 cell lines, very intense, but heterogeneous surface staining was seen. In the A549 lung and DU145 prostate lines, moderate surface staining for nucleolin was seen. In the permeabilised AML and solid tumour cells, all lines showed similar levels of homogeneous, intense intracellular staining, as expected for an ubiquitous intracellular and intranuclear protein. In SRB in vitro cytotoxicity assays, AS1411 resulted in cell death with an EC50 of 0.6 to 5 μM; the MV4-11 line was most sensitive; the EC50 for solid tumour cell lines ranged between 2 and 8 μM. Since exposure of cells to AS1411 results in S-phase cell cycle arrest; we postulated that combination of AS1411 with S-phase dependent chemotherapeutic agents may result in increased cytotoxicity and synergistic activity. An early study of the combination of AS1411 with cytosine arabinoside (AraC) indicates that there is a synergistic effect between the two drugs; the data also suggest that the synergy may be schedule dependent and requires prior exposure to AS1411 before treatment with AraC. The distribution of the aptamer in the mouse was examined using tritiated-AS1411. A total dose of 158 kBq of 3H-AS1411 was given by intravenous injection to mice bearing solid human tumour xenografts and samples of tumours and normal tissue taken between 1 hour and 4 days post injection. Approximately 1.6 to 3.1% of the injected dose was measured in the bone and bone marrow 24 hours after dosing. The ratio of the amount of 3H-AS1411 in the bone marrow to that in the blood ranged between 1.3 and 3.7 throughout the 4-day sampling period. In conclusion, we have demonstrated that nucleolin appears to be intensely expressed on the surface of AML cell lines. AML cell lines are killed by treatment with AS1411 and synergistic cytotoxicity is seen when the AS1411 is combined with AraC. In addition, the bone marrow compartment is accessible to AS1411. Taken together these findings support investigating the anti-cancer effects of AS1411 in AML patients either alone or in combination with standard AraC therapy.

Targeting the mTOR Pathway Suppresses the Growth of Acute Myeloid Leukemia (AML) Progenitors and Enhances Arsenic Trioxide Induced Antileukemic Responses.

-Background: A limiting factor for the use of arsenic trioxide (As2O3) in the treatment of non-APL subtypes of AML is the requirement of very high concentrations for the induction of leukemic cell death. The mammalian target of rapamycin (mTOR) is a key cellular regulator of multiple signaling events that control initiation of mRNA translation in leukemia cells. We examined the effects of As2O3 on mTOR-dependent cellular pathways in AML cells and determined whether modulation of the mTOR pathway sensitizes them to the antileukemic properties of As2O3.Methods: The human AML cell lines KG1, U937, or MM6 were treated with increasing concentrations of As2O3. Activation of p70 S6 kinase (p70S6K) was evaluated by immune-complex kinase assays in anti-p70S6K immunoprecipitates, while phosphorylation of various downstream mTOR effectors was assessed by immunoblotting with specific antibodies against phosphorylated S6 ribosomal protein (rpS6) and the translational repressor 4E-BP1. MTT assays were used to assess proliferation of the various cell lines, while apoptosis was determined by annexin V-propidium iodide staining. Finally, bone marrow mononuclear cells from patients with AML were used in clonogenic assays in methylcellulose to assess the effects of As2O3 and/or rapamycin on leukemic progenitor cell growth.Results: Treatment of AML cells with As2O3 resulted in activation of the p70 S6K and phosphorylation of its downstream effector, rpS6, on Ser240/244, and such events were mTOR-dependent. In addition, the translational repressor 4E-BP1 was phosphorylated on Thr37/46 and Thr70, sites whose phosphorylation is required for 4E-BP1 de-activation and dissociation from eIF4E. Pharmacological inhibition of mTOR activity blocked the growth of AML cell lines as determined by MTT assays, while the addition of As2O3 to the cultures further enhanced the inhibitory effects of rapamycin. On the other hand, AS2O3-dependent induction of apoptosis was not enhanced by rapamycin in AML cells, suggesting selective effects on cell cycle and growth inhibition. Rapamycin also inhibited leukemic CFU-GM progenitor colony formation from 6 AML patients and enhanced the suppressive effects of AS2O3 on leukemic progenitor cell growth.Conclusions: Paradoxically, As2O3 activates the pro-growth and pro-survival mTOR pathway in AML cells. Such activation appears to occur in a negative feedback regulatory manner to compensate for AS2O3 responses. Thus, combinations of AS2O3 with mTOR inhibitors may be an effective approach to overcome AS2O3-resistance in AML cells.