Acute Myelogenous Leukemia Cell Lines Research Articles

Abstract C170: The combination of statins and dipyridamole is effective preclinically in AML, MM, and breast cancer.

Abstract Statins are drugs that have been utilized for years to treat hyperlipidemia through inhibition of the rate-limiting enzyme of the mevalonate (MVA) pathway, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR). Preclinical evidence has demonstrated statins to possess anti-cancer properties against a wide range of tumors but not normal cells. Through the use of a chemical library screen, we hypothesize that the identification of compounds which potentiate the anti-cancer effects of statins will uncover novel molecular pathways and/or targets that can be exploited in combination with the MVA pathway to maximize tumor cell death. A pilot 100-compound library, composed of off-patent pharmacologically active drugs clinically used for a wide spectrum of diseases was screened in the multiple myeloma (MM) KMS11 cell line. Dipyridamole (DP), a commonly prescribed anti-platelet agent potentiated the anti-cancer effects of atorvastatin. The DP-statin combination was synergistic and capable of inducing apoptosis in a variety of acute myelogenous leukemia (AML), MM and breast cancer cell lines. The DP-statin combination also induced apoptosis in primary AML patient samples, but was not toxic to normal PBSCs. In an in vivo AML tumor model, the DP-statin combination was found to be effective at inhibiting tumor growth. DP is known to elicit numerous effects, amongst them, phosphodiesterase (PDE) inhibition. In AML cell lines, activators of the PKA pathway including other PDE inhibitors, also induced apoptosis in combination with statins similar to DP. Interestingly, the DP-statin combination prevented the increase of HMGCR, which occurs following statin treatment as part of a classic feedback response. Further mechanistic investigations to determine how DP potentiates statin-induced apoptosis are underway. As both statins and DP are pre-approved for use in humans, off-patent, and readily available, they have the potential to directly impact patient care. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C170.

Targeting AML through DR4 with a novel variant of rhTRAIL

Despite progress in the treatment of acute myelogenous leukaemia (AML) the outcome often remains poor. Tumour necrosis factor related apoptosis-inducing ligand (TRAIL) is a promising therapeutic agent in many different types of tumours, but AML cells are relatively insensitive to TRAIL-induced apoptosis. Here we show that TRAIL-induced apoptosis in AML cells is predominantly mediated by death receptor 4 (DR4) and not DR5. Therefore, we constructed a variant of TRAIL (rhTRAIL-C3) that is a strong inducer of DR4-mediated apoptosis. TRAIL-C3 demonstrated much stronger pro-apoptotic activity than wild-type (WT) TRAIL in a panel of AML cell lines as well as in primary AML blasts. The higher pro-apoptotic potential was further enhanced when the TRAIL mutant was used in combination with BMS-345541, a selective inhibitor of inhibitor-κB kinases. It illustrates that combination of this TRAIL variant with chemotherapeutics or other targeted agents can kill AML with high efficacy. This may represent a major advantage over the currently used therapies that have serious toxic side effects. The high efficacy of rhTRAIL-C3 containing therapies may enable the use of lower drug doses to reduce the toxic side effects and improve patient outcome. Our findings suggest that the rational design of TRAIL variants that target DR4 potentiate the death-inducing activity of TRAIL and offer a novel therapeutic strategy for the treatment of AML.

Personalized medicine for acute myelogenous leukemia—At the entrance gate

With the advent of molecularly targeted therapies in the oncology clinic, previously fatal blood cancers have now become chronic diseases. Imatinib, a small molecule tyrosine kinase inhibitor that targets the BCR-ABL fusion protein, dramatically extends survival for patients with chronic myelogenous leukemia and has opened the flood gates for modern ‘‘personalized cancer medicine’’. Rituximab, a monoclonal antibody with affinity for the CD20 antigen of B-lymphocytes, in an analogous fashion has revolutionized treatment for patients with B-cell lymphomas. Does this paradigm, a rather commonplace approach for the treatment of hematologic malignancies, apply also to acute myelogenous leukemia (AML)? This is an exciting time to be involved in AML research, and an explosion of new basic science discoveries has formed a springboard for some brand new personalized treatment concepts that this commentary will serve to outline. Targeted Therapy

Proteasome Inhibition in Myelodysplastic Syndromes and Acute Myelogenous Leukemia Cell Lines

In this work, effects of bortezomib on apoptosis, clonal progenitor growth, cytokine production, and NF-κB expression in patients with MDS with cytopenias requiring transfusion support are examined. Bortezomib increased apoptosis in marrow mononuclear cells but had no effects on CFU-GM, BFU-E, or CFU-L content. No consistent effects on NF-κB activation in vivo were noted. To further define the role of bortezomib in AML and MDS, we examined it in combination with several targeted agents and chemotherapeutic agents in vitro. Combinations with arsenic trioxide, sorafenib, and cytarabine demonstrated synergistic in vitro effects in AML cell lines.

A genistein derivative, ITB-301, induces microtubule depolymerization and mitotic arrest in multidrug-resistant ovarian cancer

PurposeTo investigate the mechanistic basis of the anti-tumor effect of the compound ITB-301.MethodsChemical modifications of genistein have been introduced to improve its solubility and efficacy. The anti-tumor effects were tested in ovarian cancer cells using proliferation assays, cell cycle analysis, immunofluorescence, and microscopy.ResultsIn this work, we show that a unique glycoside of genistein, ITB-301, inhibits the proliferation of SKOv3 ovarian cancer cells. We found that the 50% growth inhibitory concentration of ITB-301 in SKOv3 cells was 0.5 μM. Similar results were obtained in breast cancer, ovarian cancer, and acute myelogenous leukemia cell lines. ITB-301 induced significant time- and dose-dependent microtubule depolymerization. This depolymerization resulted in mitotic arrest and inhibited proliferation in all ovarian cancer cell lines examined including SKOv3, ES2, HeyA8, and HeyA8-MDR cells. The cytotoxic effect of ITB-301 was dependent on its induction of mitotic arrest as siRNA-mediated depletion of BUBR1 significantly reduced the cytotoxic effects of ITB-301, even at a concentration of 10 μM. Importantly, efflux-mediated drug resistance did not alter the cytotoxic effect of ITB-301 in two independent cancer cell models of drug resistance.ConclusionThese results identify ITB-301 as a novel anti-tubulin agent that could be used in cancers that are multidrug resistant. We propose a structural model for the binding of ITB-301 to α- and β-tubulin dimers on the basis of molecular docking simulations. This model provides a rationale for future work aimed at designing of more potent analogs.Electronic supplementary materialThe online version of this article (doi:10.1007/s00280-011-1575-2) contains supplementary material, which is available to authorized users.

What Role for Angiogenesis in Childhood Acute Lymphoblastic Leukaemia?

The role of angiogenesis in acute leukaemia has been discussed since the cloning of the gene of vascular endothelial growth factor (VEGF) from the acute myelogenous leukemia cell line (HL60) and, thereafter, when the first studies reported increased bone marrow vascularity and elevation of angiogenic cytokines in acute lymphoblastic leukaemia (ALL). VEGF and basic fibroblast growth factor (bFGF) are the major proangiogenic cytokines that have been studied, and evaluation of their prognostic impact in childhood ALL has been reported in several studies, though with controversial results. The antiangiogenic response, contributing to the angiogenic balance, has scarcely been reported. The origin of the factors, their prognostic value, and their relevance as good markers of what really happens in the bone marrow are discussed in this paper. The place of antiangiogenic drugs in ALL has to be defined in the global treatment strategy.

MicroSPECT/CT Imaging of Human Leukemia Engraftment In NOD-Scid Mice Using [111In]-Labeled 7G3 Anti-CD123 Antibodies

AbstractAbstract 968Radioimmunotherapy (RIT) of acute myelogenous leukemia (AML) is a promising strategy that could improve the long-term outcome of patients. A limitation of current therapies is their inability to eradicate the leukemic stem cell (LSC), which is responsible for causing and propagating the disease and the failure to achieve long-term survival. Monoclonal antibody (mAb) 7G3 recognizes CD123 (IL-3R α) in the absence of CD131 (IL-3R β) a LSC characteristic but not of normal hematopoietic stem cells. Our laboratory is developing 111In-labeled 7G3 modified with nuclear localization sequence (NLS) peptides for Auger electron RIT of AML aimed at the LSC. These electrons have only nanometer-micrometer range but are lethal when released inside the cell nucleus. Our objective was to examine the ability of 111In-NLS-7G3 to localize at sites of AML engraftment in NOD-scid mice taking advantage of the concurrent gamma emissions of 111In for microSPECT/CT imaging. These properties were examined in NOD-scid mice engrafted with CD123+/CD131- AML cell lines which avoids the challenges of engrafting primary AML specimens including their variability in engraftment efficiency and heterogeneity in CD123 expression. Alternatively, mice bearing s.c. CD123-transfected Raji (Raji-CD123) tumors were studied. 7G3 was reacted with sulfo-SMCC to introduce maleimide groups for conjugation to NLS peptides (CGYGPKKKRKVGG) then directly derivatized with DTPA for complexing 111In. The pharmacokinetics (PK) of elimination from the blood of 111In-NLS-7G3 intact IgG2a and F(ab')2 fragments were studied in NOD-scid and Balb/c mice. The effect of pre-administration of a 10-fold excess of unlabeled BM4 IgG2a or IgG1 irrelevant mAbs on the PK was assessed. Imaging and biodistribution studies were performed at 48 to 72 h p.i. of 111In-NLS-7G3 IgG2a and F(ab')2 in AML-engrafted mice. Sites of engraftment visualized on the images were analyzed for AML by flow cytometry. PK studies revealed an unusually rapid elimination of 111In-NLS-7G3 IgG2a but not F(ab')2 in NOD-scid compared to Balb/c mice with blood radioactivity levels at 18 h 4-fold lower (3.9 ± 0.9 vs. 17.0 ± 4.4% i.d./ml, respectively). This rapid elimination prevented uptake into Raji-CD123 xenografts and did not allow their visualization by microSPECT/CT. The elimination of 111In-NLS-7G3 F(ab')2 was not significantly different in NOD-scid or Balb/c mice (0.60 ± 0.17 vs. 0.87 ± 0.06 %i.d./ml, respectively at 72 h p.i.). Pre-administration of unlabeled BM4 improved the retention of 111In-NLS-7G3 in the blood by 3.2-fold and allowed for high uptake (14.7 ± 2.8 % i.d./g) and imaging of tumors. Xenografts also had high uptake (7.2 ± 3.8 % i.d./g) and imaged with 111In-NLS-7G3 F(ab')2. AML-3, -4 and -5 cells engrafted into bone marrow (BM) and NOD-scid mice assessed by the proportion of CD45+ cells (12%, 0.5% and 63%, respectively). CD45+ cells in the spleen for AML-5 engraftment was 83%. Uptake of 111In-NLS-7G3 F(ab')2 in the spleen and BM of AML-3 engrafted mice were 5-fold and 1.5-fold higher than 111In-NLS-BM4 control F(ab')2. The spleen was visualized by microSPECT/CT in engrafted mice receiving 111In-NLS-7G3 F(ab')2 but not with 111In-NLS-BM4 F(ab')2. In AML-3 engrafted mice imaged with 111In-NLS-7G3 IgG2a, multiple intense foci of radioactivity were noted in the spine, pelvis and femur, representing leukemia in the marrow. Extramedullary (EM) disease was also imaged in the brain and lymph nodes. Uptake in the femur was 2-fold higher in engrafted mice compared to non-engrafted mice. In AML-5 engrafted mice, outgrowths of AML at the femoral heads was imaged with 111In-NLS-7G3 IgG2a following pre-administration of an excess of unlabeled BM4. No visualization of these AML growths was noted for mice receiving 111In-NLS-BM4. We conclude that microSPECT/CT allowed sensitive detection of the specific uptake of 111In-NLS-7G3 and its F(ab')2 fragments in AML engrafted mice. PK and Raji-CD123 xenograft microSPECT/CT studies identified a tumor delivery barrier. A robust AML engraftment model was established with cells displaying the CD123+/CD131- phenotype. These results are promising for preclinical evaluation of 111In-NLS-7G3 for Auger electron RIT of AML and suggest that imaging is a useful tool to assess engraftment in NOD-scid mice as well as for evaluating response to 111In-NLS-7G3. Preliminary results of studies to examine 111In-NLS-7G3 for RIT of AML will also be presented. Disclosures:No relevant conflicts of interest to declare.

Role of Protein Disulfide Isomerase (PDI) In the Expression of Tissue Factor (TF) Procoagulant Activity on Apoptotic Myeloblasts

AbstractAbstract 152Disseminated intravascular coagulation (DIC) is a common and potentially life-threatening complication of acute myelogenous leukemia (AML). Although various components of the procoagulant and fibrinolytic systems have been implicated in its pathogenesis, aberrant expression of TF appears to be critically involved. Despite the need for more specific hemostatic agents, the molecular pathways underlying the expression of TF procoagulant activity (PCA) in AML remain obscure. In this study, we sought to identify determinants for systemic coagulation activation in a prospective cohort of 50 patients with newly diagnosed AML and conducted a series of in-vitro experiments to further characterize TF PCA regulation on myeloblasts in light of recently provided evidence. AML patients had significantly elevated plasma D-dimer and TF-specific PCA of isolated peripheral blood mononuclear cells (PB-MNCs) compared to healthy controls (n=10) and patients with ALL (n=5) or MDS (n=7), demonstrating specific upregulation of the TF-dependent coagulation pathway. In the AML cohort, TF PCA of PB-MNCs was 30-fold higher and TF+ plasma microparticles were more frequently detectable in patients with hypofibrinogenemia and a thrombohemorrhagic syndrome (n=8) as compared to patients with normal fibrinogen levels (n=42), providing further evidence that TF over-expression by myeloblasts is indeed a determinant for DIC decompensation. Physical cell disruption resulted in a 3-fold increase in TF PCA of PB-MNCs, indicating that a significant proportion of TF was non-coagulant (i.e. cryptic) on unperturbed cells. In patients with AML, TF PCA of PB-MNCs, absolute numbers of circulating blasts and lactate dehydrogenase (LDH) serum levels, a surrogate marker for leukemic cell turnover, were significantly correlated with plasma D-dimer, suggesting that membrane alterations associated with apoptosis/necrosis played a crucial role in myeloblast TF PCA expression. Extracellular PDI, which is abundantly expressed in the endoplasmic reticulum, has recently been implicated in TF regulation via its oxidoreductase and chaperone activity. In monoblastic THP1 cells harvested at varying time points during maintenance culture (n=5), we found a significant correlation between membrane PDI expression, as assessed by flow cytometry, and cell-associated TF PCA (r=0.93). Analysis of annexin V-FITC binding revealed that PDI was predominantly expressed on apoptotic monoblasts. Treatment of THP1 cells for 24 h with 1 μ M daunorubicin (DNR), a cytotoxic drug commonly used in AML therapy, induced >80% apoptosis and increased cellular TF PCA 8 ± 3-fold (n=8) independent of de-novo protein synthesis. Consistently, apoptotic cells stained extensively positive for PDI by flow cytometry. DNR-induced TF PCA was inhibited by 70 ± 6% (n=8) when THP1 cells were coincubated with RL90, an inhibitory PDI monoclonal antibody previously shown to diminish fibrin and platelet deposition in murine models of micro- and macrovascular thrombosis. Importantly, RL90 had no effect when added to THP1 cells at the end of the 24-h culture period, indicating specific effects on the expression of cellular TF PCA rather than the coagulation reaction itself. Similarly, coincubation of DNR-treated THP1 cells with the PDI inhibitor bacitracin dose-dependently (1-5 mM) inhibited TF activation by up to 93 ± 10% (n=3), while only a 30 ± 16% inhibition was observed when bacitracin was added at the end of the culture period. Virtually identical results were obtained with the AML cell lines, HL60 and U937. Furthermore, ex-vivo treatment of isolated myeloblasts from a patient with acute promyelocytic leukemia and decompensated DIC with 1 μ M DNR for 24 h induced >90% apoptosis and increased cellular TF PCA 5-fold. Extensive PDI staining was observed on apoptotic myeloblasts, and coincubation with bacitracin (5 mM) completely inhibited DNR-induced upregulation of TF PCA while not affecting baseline TF PCA expression. Finally, treatment with DNR resulted in the leakage of both LDH and PDI into the culture supernatants of AML cell lines, and soluble PDI antigen was detectable in the plasma samples of AML patients with the highest LDH serum levels. In summary, our findings suggest that, in addition to negatively charged phospholipids, surface-expressed and/or released PDI may be required for the efficient expression of TF-specific PCA on apoptotic myeloblasts. Disclosures:No relevant conflicts of interest to declare.

Single Agent Activity of the Cyclin-Dependent Kinase (CDK) Inhibitor Dinaciclib (SCH 727965) In Acute Myeloid and Lymphoid Leukemia Cells

AbstractAbstract 3981Dinaciclib (SCH 727965) is a selective and potent inhibitor of CDK 1, 2, 5 and 9 (IC50 < 5 nM) that has demonstrated in vitro and in vivo anti-tumor activity against a variety of tumor cell lines and human tumor xenograft models. The concentration of dinaciclib required to achieve these effects (< 100 nM) is achieved in clinical trials, and dinaciclib was found to have a more favorable therapeutic index, in preclinical murine models, than other CDK inhibitors. We have studied the effect of dinaciclib on human acute myelogenous (AML) and acute lymphoblastic (ALL) leukemia cell lines, including HL-60, K562 and Molt-4, and primary leukemia cells.Dose response curves (0.0004-10 μM) were generated for different exposure times (2, 24 and 72 h), and data from cell proliferation assay (WST-1) were used to calculate the IC50 values. Short 2 h exposure to dinaciclib followed by 24 h culture without drug resulted in different responses between the cell lines (IC50 values of 0.13 μM, 2.17 μM and ND; and viability at 10 μM 62%, 76% and 95%, for HL-60, Molt-4, and K562, respectively). With longer exposure times (24 and 72 h), the IC50 was similar between the cell lines (IC50 24 h values of 0.017, 0.015, and 0.019 μM for HL-60, Molt-4, and K562, respectively). However, even in the presence of the highest drug concentration tested (10 μM), approximately 5–25% of cells remained metabolically active after 24 h culture, and in a colony forming assay were able to proliferate and form colonies after removal of the drug. Longer 72 h exposure to dinaciclib (0.2-10 μM) completely inhibited cell proliferation in all cell lines and prevented colony formation.Next, we examined the effect of dinaciclib (2-200 nM) on cell cycle in HL-60 and K562 cells (2, 6, 9, 24 h). While lower drug concentrations and shorter exposures resulted in a minor increase in the proportion of cells in the G2 phase, a considerable increase of cells in the sub-G1 phase was observed with prolonged exposures and higher drug concentrations, most prominently in HL-60 cells (4h 200 nM 38%; 6h 20 nM 53% or 200 nM 71%, and 24 h 20 nM 84%), which is consistent with cell viability assay data. These findings were also confirmed by Annexin V/PI staining. To characterize the molecular mechanisms behind the induction of cell cycle arrest and apoptosis by dinaciclib, we measured the changes in protein expression of Mcl-1, phosphorylation of retinoblastoma (Rb) protein, and cleavage of PARP by Western blotting. Dinaciclib treatment in a dose- and time-dependent manner (6 and 24 h; 10–500 nM) significantly decreased the expression of anti-apoptotic protein Mcl-1, Rb phosphorylation at Ser 811/817, and induced cleavage of the PARP protein in the three cell lines tested. For HL-60 cells, even 2 h exposure to dinaciclib was able to induce these effects when cells were examined 4 h after treatment; however, both Mcl-1 and p-Rb returned to baseline 24 h later, suggesting that the cells were able to recover. Using HL-60 cells, we were also able to demonstrate that a decrease in Mcl-1 correlates with the decrease in phosphorylation of the carboxy-terminal domain of RNA polymerase II, suggesting that dinaciclib successfully inhibits CDK9 which may lead to transcriptional down-regulation of Mcl-1. Dinaciclib treatment also down-regulated the expression of XIAP, Bcl-xl, and phosphorylation of Bad at Ser 112 (the pro-survival form of Bad), while Bak and Bax levels remained unaffected. The cleavage of PARP correlated with the activation of the caspase-3 and -9, suggesting the involvement of the intrinsic pathway of apoptosis.We confirmed our findings in primary leukemia cells. Dinaciclib was able to induce growth inhibition in all 7 primary AML samples (IC50 for 24 h exposure ranging from 0.008 to 0.017 μM) and apoptosis (Annexin V/PI staining). Treatment with dinaciclib also resulted in down-regulation of Mcl-1, cleavage of PARP, and dephosphorylation of Rb in all primary leukemia cells examined. In summary, dinaciclib potently inhibits the growth and induces apoptosis of human leukemia cells in vitro. Prolonged exposure times may be required for its maximum efficacy, and given its short half-life in humans (1.5 to 3.3 hours), this should be considered when designing the clinical studies for patients with acute leukemias. Disclosures:Sadowska:Merck & Co: Research Funding. Muvarak:Merck & Co: Research Funding. Lapidus:Merck & Co: Equity Ownership, Research Funding. Bannerji:Merck & Co: Employment, Equity Ownership. Gojo:Merck & Co.: Research Funding.

AML-Derived Microvesicles as Mediators of Cell-Cell Signaling In the Leukemic Microenvironment

AbstractAbstract 1700Directed signaling between acute myelogenous leukemia (AML) cells and the bone marrow microenvironment plays an important role in leukemia cell survival. How leukemic blasts transform the homeostatic bone marrow microenvironment is not fully explained by known ligand-receptor interactions. Microvesicles (MV) are membrane-enclosed compartments that, depending on cell specific pathways, range in diameter from 50–100 nm (exosomes) to 100–1000 nm (microparticles) and are constitutively released into circulation in many tissues. Biogenesis has been found to be upregulated in a range of cancers. We hypothesized that MV-mediated transfer of signaling components by AML blasts may contribute to intercellular signaling in the leukemic niche. In an initial screen, we characterized MV that we recovered from the culture supernatant of multiple AML-derived cell lines, as well as AML cells from patients. Live-cell imaging of primary AML blasts using the exosomal pathway-specific dye N-Rh-PE revealed that at least a portion of these vesicles could be categorized as exosomes. Consistent with the non-random incorporation of molecular cargo observed in recent studies with solid tumors, we found the protein and RNA content of MV to be distinct from cellular lysates. In a focused, candidate target approach, our experiments showed that MV contain mRNA transcripts encoding the broadly relevant AML prognostic markers FLT3-ITD, NPM1, and IGF-1R. In both cell lines and cultured primary AML cells, insulin receptor (IR) and IGF-1R mRNAs were enriched up to ∼400-fold over GAPDH in MV versus cellular fractions. The IGF-1R receptor tyrosine kinase (RTK) transduces growth and survival signals, and the IGF-I signaling pathway has recently been identified as a potentially important candidate for molecularly targeted drug therapy for AML. Corroborative evidence for a role of MV and IGF-I signaling in shaping the leukemic microenvironment comes from the detection of human IGF-1R mRNA in cDNA prepared from mouse stromal cells (OP9) co-cultured for 48 hours with primary AML cells across a cell impermeable transwell barrier. To determine if the mRNA is transferred in a biologically significant quantity, we developed a plasmid-based quantitative PCR method to measure and compare transferred transcripts to steady state IGF-1R mRNA levels in the AML cell line HL-60. Results indicated that 0.3 HL-60 cellular equivalents of the human IGF-1R transcript were detected per OP9 cell following co-culture with primary AML cells. These data imply that transfer occurs at a level that may be functionally relevant. To further explore the potential role of MV in AML-stroma signaling, experiments are underway with HL-60 cells stably transfected with a pcDNA vector encoding the human IGF1R with a C-terminal FLAG tag. These HL-60:IGF1R-FLAG cells release MV that contain the IGF1R-FLAG transcript and will serve as a model to explore the downstream intracellular RTK signaling. In conclusion, we report for the first time the production of MV containing protein and disease-specific transcripts by AML blast cells, the selective enrichment in MV of disease-relevant transcripts, and direct cell-cell transfer of leukemia cell mRNAs to bystander cells. Our findings underscore the potential role of MV in shaping the AML microenvironment through lateral transfer of RTK mRNA transcripts. We propose a role for MV during disease progression and highlight their potential as highly sensitive, cell-free, and minimally invasive AML biomarkers. Disclosures:No relevant conflicts of interest to declare.

Adamantyl-Substituted Retinoid-Related Molecules Induce Apoptosis in Human Acute Myelogenous Leukemia Cells

The adamantyl-substituted retinoid-related (ARR) compounds 3-Cl-AHPC and AHP3 induce apoptosis in vitro and in vivo in a newly established human acute myelogenous leukemia (AML) cell line, FFMA-AML, and in the established TF(v-SRC) AML cell line. FFMA-AML and TF(v-SRC) cells displayed resistance to apoptosis mediated by the standard retinoids (including trans-retinoic acid, 9-cis-retinoic acid, and the synthetic retinoid TTNPB) but showed sensitivity to apoptosis mediated by 3-Cl-AHPC- and AHP3 in vitro and in vivo as documented by poly(ADP-ribose) polymerase (PARP) cleavage and apoptosis terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling assay. 3-Cl-AHPC or AHP3 exposure in vitro resulted in decreased expression of the antiapoptotic proteins (cellular inhibitor of apoptosis 1, X-linked inhibitor of apoptosis protein) and phospho-Bad and activated the NF-κB canonical pathway. A significant prolongation of survival was observed both in nonobese diabetic severe combined immunodeficient mice carrying FFMA-AML cells and treated with either 3-Cl-AHPC or AHP3 and in severe combined immunodeficient mice carrying TF(v-SRC) cells and treated with AHP3. We have previously shown that ARRs bind to the orphan nuclear receptor small heterodimer partner (SHP) and that the expression of SHP is required for ARR-mediated apoptosis. Induced loss of SHP in these AML cells blocked 3-Cl-AHPC- and AHP3-mediated induction of apoptosis. These results support the further development of 3-Cl-AHPC and AHP3 as potential therapeutic agents in the treatment of AML patients.

Effect of thalidomide and arsenic trioxide on the release of tumor necrosis factor-α and vascular endothelial growth factor from the KG-1a human acute myelogenous leukemia cell line

Studies conducted in our lab have indicated that thalidomide cytotoxicity in the KG-1a human acute myelogenous leukemia (AML) cell line was enhanced by combining it with arsenic trioxide. The current investigation was conducted in order to evaluate the effect of thalidomide either alone or in combination with arsenic trioxide on the release of tumor necrosis factor-α (TNF-α) and vascular endothelial growth factor (VEGF) from this cell line in an attempt to clarify its possible cytotoxic mechanism(s). Human AML cell line KG-1a was used in this study. The cells were cultured for 48 h in the presence or absence of thalidomide (5 mg/l), and or arsenic trioxide (4 μM). The levels of TNF-α and VEGF in the supernatant were determined by ELISA. Results obtained indicate that the levels of TNF-α in the supernatant of KG-1a cell cultures incubated with thalidomide, arsenic trioxide, or combination were statistically lower than those observed in the supernatant of control cells (2.89, 5.07, 4.15 and 16.88 pg/ml, respectively). However, the levels of VEGF in the supernatant of thalidomide-treated cells were statistically higher than those in the supernatant of control cells (69.61 vs. 11.48 pg/l). Arsenic trioxide, whether alone or in combination with thalidomide, did not produce any statistically significant difference in the levels of VEGF as compared to the control or thalidomide-treated cell supernatant. These findings indicate that thalidomide and the arsenic trioxide inhibition of TNF-α production by KG-1a cells may play an important role in their cytotoxic effect.

Gemtuzumab ozogamicin (Mylotarg®) upregulates tissue factor expression but not secretion of inflammatory cytokines TNF-α or IL-8

Gemtuzumab ozogamicin (GO; Mylotarg ®) is an antibody-targeted chemotherapeutic agent approved for the treatment of CD33+ acute myelogenous leukemia (AML). GO is more commonly associated with the development of Sinusoidal Obstructive Syndrome (SOS) than any other chemotherapeutic agent in this patient group. Previous investigations have shown that SOS is a pro-coagulant and pro-inflammatory syndrome. Treatment of THP1 cells (CD33+ AML cell line) with GO alone, or in combination with unconjugated CD33 antibody (hP67.6), resulted in a statistically significant ( P < 0.01) increase in tissue factor (TF) expression, but HepG2 cells (CD33 hepatocyte cell line) were unaffected. Cytokine secretion was not affected by GO treatment in either cell line. These results indicate that GO exerts a pro-coagulant response in certain cell types, which may predispose individuals to developing SOS.

Abstract 2745: Expression of immunoglobulin gene in myeloblasts from patients with acute myelogenous leukemia and its distinct VKJK recombination pattern

Abstract Immunoglobulin (Ig) gene expression is normally restricted to B lymphocytes and plasma cells. Immunoglobulin gene rearrangement has been used in the diagnosis and monitoring of response to therapy in B-cell lymphoma and leukemia. However, during the last decade, our group and others have found that Ig is also expressed in many non-hematopoietic cells, especially epithelial cancer cells. Moreover, we have reported that non-hematopoietic cancer-derived Ig expression is associated with poor prognosis, though the mechanism of its function in the survival of cancer cells is not yet known. In this study, we assessed the expression of Ig heavy chain (gamma and mu) and light chain (kappa) in myeloblasts (CD33+CD19-CD138-, sorted by FACs) from 16 patients with acute myelogenous leukemia (AML) and 6 AML cell lines by RT-PCR, ELISA, and immunohistochemistry. As control groups, we also evaluated IgG, IgM and IgK transcripts in monocytes and neutrophils (sorted by FACs) from 15 patients with non-hematopoietic malignancies and 8 healthy individuals. We have found that Ig gamma chain was expressed in 6 of 14 AML patients, but in none of the 23 controls. All of the variable region sequences of AML-derived Ig gamma chain showed diversity, similar to B cells-derived Ig gamma chain. In contrast, Ig mu chain was expressed in 7 of 14 AML cases, 3 of 15 monocytes and 1 of 10 neutrophils from non-hematopoietic cancers, and 4 of 8 monocytes and 2 of 8 neutrophils from healthy controls. All of the variable region sequences of AML-derived mu chain from any individual showed monoclonal pattern, but different individuals showed different sequences. Kappa light chain was expressed in 15 of 16 AML cases, 11 of 15 monocytes and 3 of 10 neutrophils from patients with non-hematopoietic cancers, and 1 of 8 monocytes and 1 of 8 neutrophils from healthy controls. Interestingly, the AML-derived Ig kappa transcripts showed unique VKJK rearrangement pattern with several predominant VKJK usages. In particular, the VK1-5/JK1-3 recombination was observed in 93/155 of VKJK sequences from 15 AML cases. The frequent expression of Ig in AML cells and the preferential usage of certain VKJK rearrangements by AML-derived Ig kappa light chain suggest a role of Ig in AML pathogenesis and a potential target for AML therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2745.

AEE788 is a vascular endothelial growth factor receptor tyrosine kinase inhibitor with antiproliferative and proapoptotic effects in acute myeloid leukemia

Aberrant activation of tyrosine kinase receptors is frequently observed in acute myelogenous leukemia (AML). Moreover, activating mutations of the fms-like tyrosine kinase 3 (FLT3) receptor can be found in approximately 30% of patients, thereby representing one of the most frequent single genetic alterations in AML. AEE788, a novel dual receptor tyrosine kinase inhibitor of endothelial growth factor and vascular endothelial growth factor (VEGF), is being studied in several solid tumors with remarkable success. It is not known, however, about the efficacy of this inhibitor in the treatment of AML. Therefore, we investigated the effect of AEE788 in the treatment of three human AML cell lines and seven AML patient samples. Cell survival in THP-1, MOLM-13, and MV4-11 cell lines (the two last harboring the FLT3/internal tandem duplication mutation) and AML blasts incubated with 0.5 to 15 microM AEE788 were quantified. We also studied the activation of VEGF/VEGF receptors loop, FLT3, and their downstream effectors (Akt, extracellular signal-regulated kinase, signal transducers and activators of transcription 5, and nuclear factor-kappaB). Our data showed that AEE788 was a tyrosine kinase inhibitor of FLT3 activity and had antiproliferative and proapoptotic activity in AML-derived cell lines and AML blasts that presented phosphorylation of the FLT3 receptor. Consistently, in these cells AEE788 abrogated VEGF/VEGF receptors activation and the survival signaling pathways studied. Taken together, the activity of AEE788 might represent a promising new option of targeting FLT3 for the treatment of AML.

The Triterpenoid CDDO-Me Delays Murine Acute Graft-versus-Host Disease with the Preservation of Graft-versus-Tumor Effects after Allogeneic Bone Marrow Transplantation

The occurrence of acute graft-versus-host disease (aGVHD) and tumor relapse represent the two major obstacles impeding the efficacy of allogeneic bone marrow transplantation (BMT) in cancer. We have previously shown that the synthetic triterpenoid 2-cyano-3, 12-dioxooleana-1, 9-dien-28-oic acid (CDDO) can inhibit murine early aGVHD, but antitumor effects were not assessed. In the current study, we found that a new derivative of CDDO, CDDO-Me, had an increased ability to inhibit allogeneic T cell responses and induce cell death of alloreactive T cells in vitro. Administration of CDDO-Me to mice following allogeneic BMT resulted in significant and increased protection from lethal aGVHD compared to CDDO. This correlated with reduced TNF-alpha production, reduced donor T cell proliferation, and decreased adhesion molecule (alpha(4)beta(7) integrin) expression on the donor T cells. CDDO-Me was also superior to CDDO in inhibiting leukemia growth in vitro. When CDDO-Me was administered following an allogeneic BMT to leukemia-bearing mice, significant increases in survival were observed. These findings suggest that CDDO-Me is superior to CDDO in delaying aGVHD, while preserving or possibly even augmenting GVT effects.

Screening for methylation-silenced genes in acute myeloid leukemia HL-60 cell line by a quantitative proteomic approach.

To screen for new methylation association genes in HL-60 to reveal the pathogenesis of leukemia, and provide important theoretical and scientific basis for the prevention and cure of leukemia. Two-dimensional fluorescence difference gel electrophoresis (F-2D-DIGE) was performed to separate the total proteins from acute myelogenous leukemia (AML) cell line HL-60 cells with or without 5-aza-2-deoxycytidine (5-aza-2-dC) treatment. Imaging software Decyder 6.5 and PDQuest were used to detect the differential expression protein spots, and matrix-assisted laser desorption/ionizaion time-of-flight mas spectrometer (MALDI-TOF MS) was adopted to identify the differential expression proteins. F-2D-DIGE maps of 5-aza-2-dC-untreated HL-60 and-treated HL-60 cells were established. A total of 53 differential protein spots were detected, and 35 differential proteins were successfully identified. Of the identified proteins, 32 proteins were up-regulated, and 3 proteins were down-regulated in HL-60 cells after 5-aza-2-dC treatment. Thirty-five differential proteins may be associated with methylation in HL-60 cell line, which provides the important clues for epigenetic study of leukemia.

Erucylphosphohomocholine, the first intravenously applicable alkylphosphocholine, is cytotoxic to acute myelogenous leukemia cells through JNK- and PP2A-dependent mechanisms

Alkylphospholipids and alkylphosphocholines (APCs) are promising antitumor agents, which target the plasma membrane and affect multiple signal transduction networks. We investigated the therapeutic potential of erucylphosphohomocholine (ErPC3), the first intravenously applicable APC, in human acute myelogenous leukemia (AML) cells. ErPC3 was tested on AML cell lines, as well as AML primary cells. At short (6-12 h) incubation times, the drug blocked cells in G2/M phase of the cell cycle, whereas, at longer incubation times, it decreased survival and induced cell death by apoptosis. ErPC3 caused JNK 1/2 activation as well as ERK 1/2 dephosphorylation. Pharmacological inhibition of caspase-3 or a JNK 1/2 inhibitor peptide markedly reduced ErPC3 cytotoxicity. Protein phosphatase 2A downregulation by siRNA opposed ERK 1/2 dephosphorylation and blunted the cytotoxic effect of ErPC3. ErPC3 was cytotoxic to AML primary cells and reduced the clonogenic activity of CD34(+) leukemic cells. ErPC3 induced a significant apoptosis in the compartment (CD34(+) CD38(Low/Neg) CD123(+)) enriched in putative leukemia-initiating cells. This conclusion was supported by ErPC3 cytotoxicity on AML blasts showing high aldehyde dehydrogenase activity and on the side population of AML cell lines and blasts. These findings indicate that ErPC3 might be a promising therapeutic agent for the treatment of AML patients.

JTE‐607, a multiple cytokine production inhibitor, induces apoptosis accompanied by an increase in p21waf1/cip1 in acute myelogenous leukemia cells

Proinflammatory cytokines and growth factors have been thought to play crucial roles in the pathology of acute myelogenous leukemia (AML) by supporting the proliferation and survival of AML cells in an autocrine and paracrine manner, although further elucidation is required. JTE-607 was originally identified as a multiple cytokine inhibitor that suppresses production of proinflammatory cytokines from lipopolysaccharide (LPS)-stimulated peripheral blood mononuclear cells. Herein, we report that JTE-607 exhibits inhibitory activity on the growth of AML cell lines accompanying reduction of the proinflammatory cytokine and growth factor production. In in vitro studies, JTE-607 suppressed expression and production of cytokines, which are spontaneously up-regulated in AML cell lines. JTE-607 also abrogated proliferation of AML cells in a concentration range in which colony formation of normal bone marrow cells was not affected. The growth inhibition by JTE-607 was characterized by induction of cell-cycle arrest at the S-phase and apoptosis, accompanied by a decrease in c-Myc and increase in p21(waf1/cip1). In a leukemia model engrafted with U-937 cells, JTE-607 significantly prolonged survival in mice and reduced human cytokine mRNA levels in the bone marrow. These results suggest the usefulness of JTE-607 in therapeutic applications for patients with hypercytokinemia and aggressive AML cell proliferation.

Sequential CD34 cell fractionation by magnetophoresis in a magnetic dipole flow sorter.

Cell separation and fractionation based on fluorescent and magnetic labeling procedures are common tools in contemporary research. These techniques rely on binding of fluorophores or magnetic particles conjugated to antibodies to target cells. Cell surface marker expression levels within cell populations vary with progression through the cell cycle. In an earlier work we showed the reproducible magnetic fractionation (single pass) of the Jurkat cell line based on the population distribution of CD45 surface marker expression. Here we present a study on magnetic fractionation of a stem and progenitor cell (SPC) population using the established acute myelogenous leukemia cell line KG-1a as a cell model. The cells express a CD34 cell surface marker associated with the hematopoietic progenitor cell activity and the progenitor cell lineage commitment. The CD34 expression level is approximately an order of magnitude lower than that of the CD45 marker, which required further improvements of the magnetic fractionation apparatus. The cells were immunomagnetically labeled using a sandwich of anti-CD34 antibody-phycoerythrin (PE) conjugate and anti-PE magnetic nanobead and fractionated into eight components using a continuous flow dipole magnetophoresis apparatus. The CD34 marker expression distribution between sorted fractions was measured by quantitative PE flow cytometry (using QuantiBRITE PE calibration beads), and it was shown to be correlated with the cell magnetophoretic mobility distribution. A flow outlet addressing scheme based on the concept of the transport lamina thickness was used to control cell distribution between the eight outlet ports. The fractional cell distributions showed good agreement with numerical simulations of the fractionation based on the cell magnetophoretic mobility distribution in the unsorted sample.