Kasumi-1 Cells Research Articles

Gli inhibitor GANT61 causes apoptosis in myeloid leukemia cells and acts in synergy with rapamycin

Aberrant reactivation of Gli signaling has been described in a wide variety of human cancers and rapamycin can down-regulate Gli pathway in some solid tumors. In this study, we attempt to define the cytotoxic effect of Gli inhibitor on AML cells. And the regulation action of rapamycin on Gli in AML cells also has been assessed. Gli inhibitor GANT61 caused growth arrest and apoptosis in AML cells. Rapamycin decreased not only the Gli protein and mRNA expressions but also expression of the Gli-luciferase reporter in AML cells. Synergism effect between GANT61 and rapamycin was found in Kasumi-1, HL-60 and U937 cell lines. The results suggest that aberrant Gli activation is a feature of some myeloid leukemic cells and Gli activiation can be down-regulated by rapamycin.

MicroRNA-130a–mediated down-regulation of Smad4 contributes to reduced sensitivity to TGF-β1 stimulation in granulocytic precursors

AbstractSmad4 is important in the TGF-β pathway and required for transcriptional activation and inhibition of cell growth after TGF-β1 stimulation. We demonstrate that miR-130a is differentially expressed during granulopoiesis and targets Smad4 mRNA. The transcript for Smad4 is present throughout neutrophil maturation, but Smad4 protein is undetectable in the most immature cells, where miR-130a is highly expressed. Two miR-130a binding sites were identified in the 3′-untranslated region of the Smad4 mRNA. Overexpression of miR-130a in HEK293, A549, and 32Dcl3 cells repressed synthesis of Smad4 protein without affecting Smad4 mRNA level. Repression of Smad4 synthesis in a granulocytic cell line by miR-130a reduced its sensitivity to TGF-β1–induced growth inhibition. This effect was reversed by inhibiting the activity of miR-130a with an antisense probe or by expressing a Smad4 mRNA lacking miR-130a binding sites. High endogenous miR-130a and Smad4 mRNA levels and low expression of Smad4 protein were found in the t(8;21)(q22;q22) acute myelogenous leukemia–derived cell line Kasumi-1. When miR-130a was inhibited by an antisense RNA, the amount of Smad4 protein increased in Kasumi-1 cells and rendered it susceptible for TGF-β1–mediated cell growth inhibition. Our data indicate that miR-130a is involved in cell cycle regulation of granulocytic cells through engagement of Smad4 in the TGF-β pathway.

Sensitivity of acute myeloid leukemia Kasumi-1 cells to binase toxic action depends on the expression of KIT and АML1-ETO oncogenes

Some RNases selectively attack malignant cells, triggering an apoptotic response, and therefore are considered as alternative chemotherapeutic drugs. Here we studied the effects of Bacillus intermedius RNase (binase) on murine myeloid progenitor cells FDC-P1; transduced FDC-P1 cells ectopically expressing mutated human KIT N822K oncogene and/or human AML1-ETO oncogene; and human leukemia Kasumi-1 cells expressing both of these oncogenes. Expression of both KIT and AML1-ETO oncogenes makes FDC-P1 cells sensitive to the toxic effects of binase. Kasumi-1 cells were the most responsive to the toxic actions of binase among the cell lines used in this work with an IC50 value of 0.56 µM. Either blocking the functional activity of the KIT protein with imatinib or knocking-down oncogene expression using lentiviral vectors producing shRNA against AML1-ETO or KIT eliminated the sensitivity of Kasumi-1 cells to binase toxic action and promoted their survival, even in the absence of KIT-dependent proliferation and antiapoptotic pathways. Here we provide evidence that the cooperative effect of the expression of mutated KIT and AML1-ETO oncogenes is crucial for selective toxic action of binase on malignant cells. These findings can facilitate clinical applications of binase providing a useful screen based on the presence of the corresponding target oncogenes in malignant cells.

Modulation of activated oncogene c-kit expression with RNA-interference

Hyperexpression of oncogene c-kit is found in 80% patients with acute myeloid leukemia (AML). The transgenic model cell line expressing the oncogene c-kit was obtained by transduction with recombinant retrovirus. We have designed small interfering RNAs (siRNA) efficiently suppressing the expression of activated oncogene c-kit. Further small hairpin RNAs (shRNA) targeting c-kit mRNA were designed and expressed in lentiviral vectors. We report a stable reduction in c-kit expression following the introduction of shRNAs into model cells as well as Kasumi-1 cells from the patient with AML.

Ex Vivo-Expanded Vγ9Vδ2 T Cells Can Efficiently Kill Human Acute Myeloid Leukemia Cells Via Trogocytosis

Abstract 580Cellular immunotherapy of hematopoietic malignancies is regarded as one of the most promising approaches to deal with the common relapse or resistance to conventional treatments. γδT cells are innate-like lymphocytes capable of potent antitumor activity toward a variety of malignant cell types and have been applied to clinical trials with positive effects. But it still lacks basis whether these cells coule be used in the treatment of acute myeloid leukemia(AML).Here we set out to determine the potent cytotoxicity of ex vivo-expanded Vγ9Vδ2 T cells against AML cells and the underlying mechanisms. To expand human Vγ9Vδ2 T cells, peripheral blood mononuclear cells(PBMCs)from healthy donors(HD)were cultured in the presence of 2uM zoledronate together with recombinant human IL-2 ( 250 IU/ml). After culture in vitro for 12 days, 90–95% Vγ9Vδ2 T cells were detected by flow cytometry(FACS). Cell viability was determined using trypan blue exclusion and >99% cells were viable. We selected 6 AML cell lines as target cells including Kasumi-1 and HL-60 cell lines as the AML M2 type, NB4 as the AML M3 type, K562 as the AML M6 type, HL60/ADR and K562/AO2 cell lines as the chemotherapeutics-resistant AML. First, we evaluated the cytotoxic activity of Vγ9Vδ2 T cells against different AML cell lines. Cytotoxicity was measured by FACS analysis using CFSE and 7-AAD. AML cell lines above were labeled with 1uM CFSE and then incubated with Vγ9Vδ2 T cells for 4h or 8h at the effect: target (E:T) ratios of 5:1, 10:1, and 20:1. 7-AAD was added before acquisition on the FACSCalibur cytometer. The calculation of cytolytic activity was based on the degree of reduction of viable target cells (VTC) with the ability to retain CFSE and exclude 7-AAD (CFSEhigh 7-AAD−). We found Vγ9Vδ2 T cells exerted cytotoxicity on different AML cell lines in varying degrees. At an E:T ratio of 20:1, the cytotoxicity of Vγ9Vδ2 T cells on Kasumi-1, HL-60, HL-60R, NB4, K562, K562-AO2 cell lines for 4h were: 53.27%±18.43% A12.03%±18.16 A13.47%±10.36% A17%±11.01% A29.53%±12.22% A26.4%±0.71%, and for 8h could increase to 56.86%±16.92% A32.25%±6.71% A25.9%±0.99% A41.2%±8.22% A24.27%±8.51% A24.35%±13.51% respectively(P<0.05). Interestingly, Vγ9Vδ2 T cells did not kill normal allogeneic bone marrow mononuclear cells. Then we chose Kasumi-1 cell line, which was the most sensitive to Vγ9Vδ2 T cells’ cytotoxicity, as target cells for the study of mechanism below. Kasumi-1 cells were cocultured with 10 folds of Vγ9Vδ2 T cells for 24h and cell morphology was observed by phase contrast microscope. We noted the refractive index of Kasumi-1 cells was significantly decreased, some cells became smaller, shrunken and disintegrated. Also some irregular cells could be seen. It has been proposed that human Vγ9Vδ2 T cells trigger several distinct pathways for killing tumor cells, which include secretion of proinflammatory cytokines and proapoptoticmolecules or cell contact-dependent lysis. Trogocytosis has been reported to play vital roles in killing chronic myeloid leukemia by Vγ9Vδ2 T cells. To know whether Vγ9Vδ2 T cells kill the AML cells in the same way, Vγ9Vδ2 T lymphocytes were further loaded with green-CFSE and coincubated for 4h with Kasumi-1 cells, which were previously labeled with orange-DiI, to measure Vγ9Vδ2 T lymphocytes’ ability to conjugate with target cells into lytic synapses by fluorescence microscope. The result revealed that Vγ9Vδ2 T cells were significantly activated after 1h coincubation, and the target cells staying in the middle were surrounded by metatypical Vγ9Vδ2 T cell populations. When the two groups of cells were incubated for 4h, we observed Vγ9Vδ2 T lymphocytes captured the Kasumi-1 cells’ membrane fragments by direct contact. In summary, our findings indicate that ex vivo-expanded Vγ9Vδ2 T cells possess a promising cytotoxic activity against not only AML cells, but also chemotherapeutics resistant cells, especially AML M2-type cell line-Kasumi-1, with t (8,21) characteristics. We also discover that the death of target cells relies much upon cell contact with Vγ9Vδ2 T lymphocytes. The further mechanisms involved in Vγ9Vδ2 T cell-mediated cytotoxicity following direct contact and the reason of varying sensitivity still remain unclear. We will clarify all these in the future. Disclosures:No relevant conflicts of interest to declare.

Significance of Fusion Genes for Maintenance of Leukaemia

Abstract 2455 Background:MLL/AF4 and AML1/MTG8 are fusion genes most frequently found in infant acute lymphoblastic leukaemia (ALL) and acute myeloid leukaemia (AML), respectively. We have previously shown that transient siRNA mediated knock-down of MLL/AF4 and AML/MTG8 impairs proliferation and clonogenicity in vitro and causes a significant increase in median survival in a xeno-transplantation model. Aims:We investigated the role of MLL/AF4 and AML1/MTG8 in leukaemic maintenance and progression of established disease in vivo. We used an inducible lentiviral shRNA expression system to determine the effects of knockdown of MLL/AF4 in the t(4;11)-positive SEM cell line and of AML1/MTG8 in the t(8;21)-positive human leukaemic cell line Kasumi-1. In addition, to allow in vivo imaging SEM and Kasumi-1 cells were labelled with luciferase. Methods:shRNA cassettes specifically targeting the MLL/AF4 and the AML1/MTG8 fusions were cloned into the pTRIPZ vector where shRNA expression and RFP expression are both induced by doxycycline. Luciferase labelled SEM and Kasumi-1 cells were transduced with lentiviral particles, selected with puromycin and transduction efficiency was determined by quantification of RFP positive cells using flow cytometry. Experiments were initiated when more than 65% of the cell populations expressed RFP. Knockdown and expression of known targets of the fusion genes were verified at both the RNA and protein level by qPCR and western blotting, respectively. Cell growth was monitored by cell counts. Immunodeficient NSG mice were given doxycycline in the diet (625ppm) from two days prior to intrafemoral transplantations with 106 luciferase labelled SEM and Kasumi cells transduced with pTRIPZshAML1/MTG8 or pTRIPZshMLL/AF4. The food was changed every other day and disease progression was monitored using in vivo bioluminescence imaging. Results:Upon induction of shRNA expression in vitro Kasumi-1 cells transduced with pTRIPZshAML1/MTG8 showed decreased expression of AML1/MTG8 at protein and RNA levels, which correlated with impaired proliferation. Furthermore, the AML1/MTG8 knockdown resulted in decreased CD34 expression and increased levels of IGFBP7 and PRG2. Induction of shMLL/AF4 expression in SEM cells resulted in decreased expression levels of MLL/AF4 with concomitant decreased expression of HOXA7. However, the number of RFP positive SEM pTRIPZshMLL/AF4 cells decreased over time. When we investigated the in vivo consequences of fusion-gene knockdown in transplanted NSG mice we found no significant differences in overall survival. Notably, mice transplanted with pTRIPZshAML1/MTG8-transduced Kasumi-1 cells showed a lower grade of disseminated disease compared to mice transplanted with pTRIPZshMLL/AF4-transduced Kasumi-1 cells. Furthermore, tumours from these mice had significantly lower RNA and protein levels of AML1/MTG8 (p<0.05). We could not verify knockdown of MLL/AF4 in tumour cells harvested from mice transplanted with SEM pTRIPZshMLL/AF4. Interestingly, these cells showed a complete loss of RFP expression compared to SEM transduced with pTRIPZshAML1/MTG8 (p<0.05). Conclusions:Knockdown of MLL/AF4 and AML1/MTG8 in vitro using an inducible shRNA system led to decreased proliferation and affected genes associated with differentiation. However, the effects were delayed compared to transient siRNA knockdown. In vivo optical imaging is a useful tool to monitor leukaemic progression in vivo. The technique gives information about location and degree of disease dissemination in addition to the overall survival. The loss of RFP expression particularly in SEM pTRIPZshMLL/AF4 cells both in vitro and in vivo highlights the significance of MLL/AF4 in leukaemic maintenance. Further in vitro and in vivo experiments are currently ongoing with knockdown of AML1/MTG8 and MLL/AF4 in alternative cell lines and primary patient-derived material. Disclosures:No relevant conflicts of interest to declare.

The Spindle Checkpoint Protein BubR1 Is Differentially Expressed in the Lymphatic and Myeloid Lineage and Is a Determinator of Response to Spindle Poisons

Abstract 1407 Introduction:Spindle poisons disrupt the mitotic spindle thus leading to activation of the spindle assembly checkpoint (SAC). The SAC is a mitotic surveillance mechanism that can interfere with anaphase-promoting complex/cyclosome- (APC/C-) dependent proteolysis of key cell cycle regulators, such as securin and cyclin B, to delay cells at the metaphase to anaphase transition. The SAC protein BubR1 is a tumor suppressor with critical functions during mitosis and has also been shown to be important for the stablilization of cyclin B during interphase. We found this critical player to be downregulated in AML cell lines, primary AML blast populations and myeloblast-like murine 32D cells when compared to ALL cell lines and lymphoblast-like murine BaF3 cells. While myeloblast-like 32D cells are untransformed cells but also exhibit BubR1 repression we speculate that there might be a physiological role for a reduced BubR1 expression level in healthy myelopoiesis. However, in highly proliferative AML cells BubR1 repression might be a source of genetic instability due to a less efficient SAC-mediated interference with APC/C-dependent proteolysis in the presence of inaccuracies during mitosis. Methods and results:Since repression of BubR1 is known to shorten the metaphase duration, we performed live-cell imaging of leukemia cells and found myeloblastic Kasumi-1 cells, expressing histone H2-GFP, to proceed faster through mitosis as compared to lymphoblastic DG-75 cells. While DG-75 cells exhibited a stable metaphase arrest upon spindle disruption using the spindle poison nocodazole, Kasumi-1 cells showed only a transient arrest, degraded securin and cyclin B and underwent sister-chromatid separation in the absence of a functional mitotic spindle. These findings suggest that the mitotic checkpoint is unable to properly interfere with APC/C-dependent proteolysis to prevent mitotic progression in myeloblastic leukemia upon treatment with spindle poisons. By using inducible retroviral reexpression of BubR1 and its downstream effector cyclin B we could enhance the ability of Kasumi-1 cells to accumulate in mitosis upon spindle disruption. Moreover, restoration of BubR1 led to higher cyclin B levels. Live-cell imaging analyses of Kasumi-1 cells, which expressed doxycyclin-inducible BubR1, revealed a prolonged metaphase, suggesting a more stringent control by the mitotic checkpoint when BubR1 expression is restored. Prolonged metaphase delays were also detected after reexpression of BubR1 when we challenged the cells with lower doses of spindle poisons suggesting that BubR1 is an important sensitizer for antimitotic therapies. Therefore, our finding of low BubR1 expression in AML provides an explanation for the poor response of myeloid leukemia to spindle poisons as compared to lymphoblastic leukemia. BubR1 has also been reported to be inactivated through promoter hypermethylation in various malignancies. The existence of a CpG island in the upstream region of the Bub1b locus (BubR1 coding sequence) tempted us to treat Kasumi-1 cells with the demethylating agent decitabine. Promotor demethylation led to an upregulation of BubR1 in mitotic AML cells providing evidence that BubR1 is a druggable target to enhance mitotic surveillance in AML cells. Conclusions:Because mitotic therapies are widely used in the treatment of different malignancies, a further understanding of these processes might lead to a better understanding of cancer biology and improved therapeutic approaches. Disclosures:No relevant conflicts of interest to declare.

Targeted Delivery of MicroRNA-29b by Nanoparticles Provides Antileukemic Activity and Increases Sensitivity to the Hypomethylating Agent Decitabine (DAC) in Acute Myeloid Leukemia (AML)

Abstract 81Despite advances in our understanding of disease mechanisms the outcome of the majority of AML patients (pts) remains poor. Epigenetic gene silencing by DNA hypermethylation has been shown to contribute to AML. Recently we demonstrated promising results of treatment with the hypomethylating agent DAC in older AML pts. Pts with higher miR-29b expression were more likely to respond to DAC. MiR-29b targets DNA methyltransferases (DNMTs; i.e. DNMT1, DNMT3A & DNMT3B) and has hypomethylating and tumor suppressor activity in AML. Thus uncovering a method to increase intracellular miR-29b and sensitize AML blasts to DAC may be of therapeutic value. Free synthetic miRs are degraded in bio-fluid and have very limited cellular uptake. To overcome these limitations, we developed a novel non-viral delivery system for synthetic miRs. Polyethylenimine was used to capture synthetic miRs & form a polyplex core. An outside layer of lipid components, 1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine, Linoleic acid & 1,2-Dimyristoyl-sn-glycerol, methoxypolyethylene Glycol (MW=2,000) was used to protect the miRs from degradation & clearance and facilitate their uptake. Using this approach we produced nanoparticles (NPs) encapsulating 0.4 μM synthetic miR-29b precursors (pM29b) or scramble controls (sc) [both Ambion]. Since AML blasts and cell lines overexpress the transferrin receptor, we conjugated the NPs with transferrin (Tf) to increase the uptake by targeted delivery. To evaluate the efficiency of NP uptake we encapsulated FAM-fluorescent labeled-pM29b and treated AML cell lines (i.e. Kasumi-1 & MV4-11). Tf-conjugated NP (TfNP) treated cells showed the highest uptake by confocal microscopy and flow-cytometry (mean fluorescence intensity (MFI): 21.0 & 22.9 for Kasumi-1 & MV4-11 respectively) compared to non-Tf-conjugated NPs (NP; MFI: 8.3 & 13.0). To evaluate if pM29b was processed into its mature form we measured levels of mature miR-29b in MV4-11 & Kasumi-1 by qPCR after treatment with TfNP encapsulating pM29b (TfNP29b) or NP encapsulating pM29b (NP29b). At 48h mature miR-29b increased 2000 and 2100 fold after treatment with TfNP29b but only 20 and 50 fold following treatment with NP29b in MV4-11 & Kasumi-1 respectively, compared to untreated or TfNPsc treated cells. Thus TfNPs were 40–100 times more efficient than NPs in increasing levels of miR-29b. Our observations were validated in primary blasts from 3 AML pts. At 48h miR-29b levels increase on average 400 fold in TfNP29b vs TfNPsc treated blasts. The levels of an unrelated miR (i.e. miR-140) remained unchanged in all our experiments. These results demonstrate that pM29b is efficiently delivered and processed into its mature form after TfNP delivery, leading to a specific increase of miR-29b in AML cell lines & primary blasts with otherwise low endogenous miR-29b expression. Downregulation of the miR-29b targets DNMT1, DNMT3A, and DNMT3B on RNA & protein level, determined by qPCR and western blotting respectively, was observed in both cell lines & primary AML samples at 48 hours following TfNP29b compared to TfNPsc treatment. The observed fold reduction on RNA level for DNMT1 was 0.63, 0.53 & 0.63, for DNMT3A 0.48, 0.53 & 0.58, and for DNMT3B 0.60, 0.73 & 0.53 in treated MV4-11, Kasumi-1 cells & pts blasts (mean, n=3), respectively. Increased miR-29b, following TfNP29b treatment resulted in antileukemic activity. Treatment with TfNP29b compared to TfNPsc led to 30% & 35% reduction in proliferation at day 5 and 50% & 64% decreased colony forming ability (scored after two weeks) in MV4-11 & Kasumi-1 cells, respectively. To investigate a possible sensitizing effect of miR-29b to DAC treatment, we treated MV4-11 cells and primary AML blasts with 2.5 uM DAC, which is comparable to concentrations achievable in treated AML pts. 72 hours after DAC treatment, decreased viability (measured by MTS assays) was observed in cells treated with TfNP29b (for 48h) followed by DAC compared with those treated with TfNPsc followed by DAC: 36% vs 53% (p<0.001) for MV4-11 cells and 63% vs 73% (p<0.01) for primary pts blasts. In conclusion, we report an effective nanoparticle-based delivery system for miRs. Our delivery system for pM29b showed in vitro antileukemic activity and improved response to DAC in AML cells, demonstrating that miR-29b is not only a predictor for clinical response to DAC, but may also functions as a novel therapeutic, sensitizer to hypomethylating agents in AML. Disclosures:No relevant conflicts of interest to declare.

Mir-130a-Mediated Downregulation of SMAD4 Contributes to Reduced Sensitivity to TGFβ Stimulation in Promyelocytic Cells,

Abstract 3383MicroRNAs (miRNA) are noncoding RNA molecules that regulate the synthesis of proteins and, if dysregulated, can result in development of various form of cancers. We have found that miR-130a is highly expressed in immature proliferating granulocytic precursors. In more mature granulocytes the miR-130a expression is significant lower. In acute myeloid leukemia the granulocyte precursors have lost the ability to undergo terminal maturation, leading to accumulation of non-functional, immature granulocytes (myeloblasts). We hypothesize that a sustained high expression of miR-130a during granulopoiesis may sustain continuous cell proliferation. TGF-β is a strong inhibitor of cell proliferation and lack of TGF-β expression is associated with various form of cancer. Smad4 is an essential compound in the TGF-β signaling pathway. Using microRNA target-prediction software, we identified Smad4 as a putative target for miR-130a. This was confirmed experimentally by demonstrating that transient overexpression of miR-130a results in reduction in the amount of Smad4 protein. Luciferase reporter constructs with the 3‘-UTR of Smad4 also respond to miR-130a – an effect that is abolished by point mutations in the miRNA–binding site. In agreement, we observed that stable overexpression of miR-130a in a granulocytic cell line reduces the level of Smad4 protein, and render the cells less sensitive to TGF-β-induced growth inhibition. This was also confirmed with cell cycles analysis. Furthermore, the effect was diminished when transfecting the same clones with SMAD4 lacking the 3-‘UTR. In line with our hypothesis, the most immature granulocyte precursors demonstrate the highest expression of miR-130a is highest, and the lowest expression of Smad4 protein. As the granulocyte precursors mature, the expression of miR-130a decreases dramatically whereas the level of Smad4 protein expression increases demonstrating inverse correlation between miR-130a and Smad4 protein. The level of Smad4 mRNA is comparable at all stages of granulopoiesis. High miR-130a levels and low or no expression of Smad4 was found in primary cells from patients with acute myeloid leukemia and in a cell line (Kasumi-1) with the t(8;21)(q22;q22) chromosomal translocation. The level of Smad4 increased in Kasumi-1 cells when the endogenous level of miR-130a was inhibited by anti-miR-130a LNA. Our data indicate that miR-130a is involved in cell cycle regulation of normal and malignant granulocytic cells through engagement of Smad4 in the TGF-β-pathway. Grant acknowledgment: Lundbeck foundation, Carlsberg foundation, Swedish Research Council. Disclosures:No relevant conflicts of interest to declare.

The Angiogenic Factor Angiopoietin-1 Is Regulated by the Acute Myeloid Leukemia Fusion Protein AML1/ETO

Abstract 2426 BACKGROUNDThe chromosomal rearrangement t(8;21)(q22;q22) encodes the fusion gene AML1/ETO, which is the most common translocation in acute myeloid leukemia (AML). It has an incidence of approximately 15% and a favourable prognosis in comparison to other AML subtypes. Dysregulated angiogenesis in the bone marrow niche environment is predicted to have an important role in leukemia pathogenesis, and several factors have been implicated in this process. Angiopoietin-1 (ANGPT1) is a cytokine involved in hematopoietic stem cell quiescence and regulation of microvessel density within the bone marrow, as well as having a role in transendothelial migration. Moreover, ANGPT1 is upregulated in leukemic blast cells from AML patients. In this study we investigated the role of AML1/ETO as a regulator of ANGPT1 expression, as well as functional implications of ANGPT1 in AML1/ETO-positive AML. METHODSIn order to investigate putative AML1/ETO-dependent regulation of ANGPT1, we performed gain-of-function studies using lentiviral gene transfer to ectopically express AML1/ETO in HL-60 and U937 AML cell lines. We also depleted AML1/ETO in the t(8;21)-positive AML cell line Kasumi-1 using fusion gene specific siRNA. Additionally, the functional role of ANGPT1 was studied using targeted RNAi in Kasumi-1 cells. Transcript expression of AML1/ETO and ANGPT1 was analysed by quantitative Real-Time PCR (qRT-PCR) and AML1/ETO protein expression was quantified by western blotting. Angiopoietin-1 protein secretion was determined using enzyme-linked immunosorbent assay (ELISA). We also utilised Matrigel transwell assays to test the effect of ANGPT1 downregulation on the invasive and migratory properties of Kasumi-1. RESULTSIn HL-60 and U937 transduced with AML1/ETO, we observed an up to 280 fold increase in ANGPT1 mRNA transcript levels as measured by qRT-PCR, which correlated with an increase in secreted Angiopoietin-1 protein. Conversely, siRNA-mediated AML1/ETO depletion in Kasumi-1 cells significantly decreased ANGPT1 transcript and protein levels after a single electroporation. After three serial electroporations with siRNA, AML1/ETO transcript levels were reduced by 85%, with a concomitant decline in ANGPT1 transcript (>99%) and secreted protein. Preliminary data suggest siRNA targeting of ANGPT1 in Kasumi-1 decreases the invasive ability of these cells, causing a ≥50% reduction in invasion when compared to controls. CONCLUSIONSWe have identified a strong correlation between AML1/ETO and ANGPT1 expression, whereby a reduction of AML1/ETO results in a substantial reduction of ANGPT1. Similarly, the introduction of AML1/ETO into myeloid cell lines results in a large upregulation of ANGPT1. Preliminary evidence suggests that a reduction of ANGPT1 reduces the invasive and migratory potential of Kasumi-1. This could have major functional consequences in the bone marrow niche with regards to understanding the AML stem cell and its interaction with the niche environment as well as providing insight into how leukemic cells in the circulation might interact with the vasculature. Disclosures:No relevant conflicts of interest to declare.

Pretreatment of Low-Dose Decitabine to Leukemia Cells Markedly Enhances the Cytotoxicity of Gemtuzumab Ozogamicin

Abstract 5000Prognosis of relapsed/refractory acute myeloid leukemia (AML) patients has been poor due to its drug resistance. Although gemtuzumab ozogamicin (GO) targets CD33 antigen, which is overexpressed on most AML cells, the efficacy of GO alone for relapsed/refractory patients remains to be unsatisfied. Therefore we aimed to improve GO effects by combination with the other drugs.GO needs target cells intact to complete several steps for its cytotoxicity: GO binding to CD33 antigen, internalization of GO with CD33 antigen, fusion of the endosome to lysosome, digestion of the linker by lysozyme and release of calicheamicin, and intercalation of activated calicheamicin to DNA. GO could exert its cytotoxicity only when these cellular steps of target cells are intact. This paradoxical mechanism prompted us to study decitabine (DAC), azacitidine (AZA) and, valproic acid (VPA), which modify epigenetic alterations in leukemia cells with modest cytotoxicity. Low-dose DAC, AZA, or VPA have been reported to differentiate AML cells, with the increased expression of myeloperoxidase and lysozyme. Differentiation of AML cells by some agents, for example G-CSF, has been reported to reduce the expression of P-glycoprotein. Thus, we hypothesized that DAC, AZA or VPA could activate the cellular functions of target cells and enhance the cytotoxicity of GO.As refractory leukemic cells, we used SKK-1 and SKM-1 cells derived from patients with leukemia progressed from myelodysplastic syndrome, and SKNO-1, KO52, and Kasumi3 cells with complex karyotypes. We also used an acute promyelocytic leukemia cell line (NB4) and an acute myelomonocytic leukemia cell line (U937).Low-dose of DAC (100nM), AZA (2μM), or VPA (1mM) showed negligible cytotoxic effects. Simultaneous administration of GO (2.5 μg/ml) and DAC did not enhance cytotoxicity of GO. In contrast, GO cytotoxicity was enhanced only when cells were pretreated with DAC prior to GO. Pretreatment with DAC for 48 hours showed maximum enhancing effects. AZA also required pretreatment for enhancing effect. It was suggested that DAC and AZA induced some cellular response which enhance the cytotoxic steps of GO during pretreatment. VPA enhanced the cytotoxicity added either simultaneously or antecedently. The enhancing effect varied in each cell line. Pretreatment with DAC showed the most prominent enhancing effects among the three agents.DAC treatment changed neither the expression of CD33 antigens nor internalization of GO. DAC enhanced GO cytotoxicity partly by both promoting intercalation of calicheamicin to DNA and suppressing the activity of a drug-efflux pump, MRP-1.DAC-enhanced GO cytotoxicity in vitro was also observed in primary leukemic blasts of four (29%) among 14 relapsed/refractory AML patients.A large phase III study about GO, S0106 trial conducted by SWOG, revealed no significant improvement either in CR rate or in LFS for de novo AML patients, whereas significant increase of early complication death, in concurrent use of chemotherapy and GO. The result of SWOG trial and the mechanism of GO cytotoxicity might suggest that concurrent use of strongly-cytotoxic agents is not the best way of combination for GO. In contrast to SWOG trial, the present study targeted relapsed/refractory AML cells, employed pretreatment of low-dose DAC, and demonstrated preclinical possibility that the combination of DAC and GO would be useful for relapsed/refractory AML. Disclosures:No relevant conflicts of interest to declare.

Statin Treatment Prevents FLT3 Glycosylation and Overcomes Resistance to FLT3 Tyrosine Kinase Inhibitors

Abstract 1421FLT3 is a receptor tyrosine kinase that is expressed on hematopoietic stem and progenitor cells where it functions in cell differentiation, survival and proliferation. It is also one of the most frequently mutated genes in acute myeloid leukemia (AML), and has thus become a target for modulation by the use of FLT3 tyrosine kinase inhibitors (TKI). Unfortunately, clinical resistance emanating via several different mechanisms has limited the potential benefit of FLT3 TKI. Some of these include FLT3 mutations that reduce drug binding, elevated FLT3 ligand (FL) levels that shift the dose-response curve and activation of parallel signaling pathways. These all pose major challenges to TKI effectiveness that must be overcome to improve patient outcomes. Since FLT3 is dependent upon N-linked glycosylation for its maturation and surface localization, we sought to determine whether statins might disrupt FLT3 signaling. Statins inhibit the mevalonate pathway and reduce levels of all ensuing end-products including dolichol, which transfers preassembled oligosaccharides onto nascent polypeptides. Here, we demonstrate by Western blotting that statins can indeed prevent complex FLT3 glycosylation, thus leading to loss of surface receptor expression. Immunofluorescence microscopy confirms a reduction in surface localization and a concomitant increase in intracellular FLT3/ITD accumulation. Interestingly, this aberrant localization was associated with increased STAT5 activation while inhibiting both MAP kinase and AKT phosphorylation. We have extended our previous findings that statins are cytotoxic to mutant FLT3 expressing cell lines to examine whether they are also able to overcome the resistance mechanisms discussed above. We show that the following mechanisms of resistance could be circumvented by fluvastatin. First, stimulation of BaF3 FLT3/ITD cells with exogenous FL at physiologic concentrations induces a three-fold increase in the IC50 for inhibition of phosphorylated FLT3/ITD by the FLT3 TKI lestaurtinib. Pretreatment with fluvastatin for 24 h eliminated surface FLT3/ITD: FL interactions and restored the potency of lestaurtinib. Secondly, resistance to another clinical FLT3 TKI, sorafenib, caused by the FLT3/ITD N676K or D835Y mutations increased the IC50 from 5 nM for BaF3/ITD cells to 30 nM and >50 nM, respectively. In contrast, both of these mutants remained just as sensitive to treatment with fluvastatin as were the BaF3/ITD cells (approx. IC50 of 0.2 μM). A third mechanism of resistance that can be circumvented by fluvastatin is the over expression and/or activation of alternative pathways that can compensate for inhibited FLT3. A model of this is rescue of BaF3 FLT3/ITD cell lines from FLT3 TKI-mediated cytotoxicity by stimulation with IL-3. When this cell line is stimulated with IL-3, fluvastatin is able to inhibit activation of STAT5, AKT and MAP kinase caused by the IL-3 receptor, as well as FLT3/ITD signaling, leading to cell death. Fluvastatin was also effective in other cell lines expressing constitutively activated FLT3 (Molm-14, MV4;11, HB1119 and SEM-K2 cells) and in other cell lines in which glycosylated transmembrane receptors drive proliferation (mutant c-Kit in Kasumi cells) but not in cells that rely upon intracellular kinases (BCR-ABL in K562 cells). Importantly, fluvastatin also reduced FLT3 glycosylation in and was cytotoxic to primary AML patient samples harboring FLT3/ITD mutations at therapeutically achievable concentrations (1 μM). Finally, fluvastatin reduced engraftment of BaF3 FLT3/ITD cells transplanted in syngeneic Balb/c mice and prolonged their survival. These results demonstrate that statins, a class of drugs already FDA approved, might be useful, either alone or in combination with a FLT3 TKI, in the management of FLT3 AML cases including those resistant to FLT3 TKI. Disclosures:Levis:Ambit Biosciences, Inc: Consultancy.

Differential Activity of Pazopanib and Imatinib Against PDGFRA-Related MPN and AML

Abstract 5153 Introduction:Pazopanib is a tyrosine kinase inhibitor with proven activity against metastatic renal cancer. Due to its target spectrum of kinases (PDGFR, KIT, and VEGFR), we sought to investigate its activity against myeloid malignancies. Methods:32D cells transduced with FIP1L1-PDGFRA or several activating PDGFRA point mutations as well as AML cell lines were analyzed for the effects of pazopanib vs. imatinib on cell growth, MTS activity, 7-AAD positivity, and clonogenic growth. Pazopanib and imatinib were purchased from LC Laboratories Woburn, MA, USA. Results:Pazopanib was found to decrease the growth of FIP1L1-PDGFRA transduced cell lines at low nanomolar concentrations (10 nM). 1000 nM doses were equally effective as 1000 nM of the PDGFR tyrosine kinase inhibitor imatinib. MTS assays confirmed that at 10 nM, pazopanib reduced cell proliferation to 28% of that of vehicle-treated control cells (DMSO 0.05%), while 100 nM of pazopanib completely abolished cell growth and suppressed MTS activity. Analysis of 7-AAD positivity using flow cytometry showed that reduction in cell growth and MTS activity was due to induction of apoptosis (17+/−0.6%, 63+/−0.5%, and 87+/−0.5%, 86+/−0.1% for 10, 100, and 1000 nM of pazopanib and 1000 nM of imatinib, respectively). Interestingly, while two PDGFRA activating point mutations (H650Q and R748G) recently identified in patients with idiopathic hypereosinophilic syndrome-type myeloproliferative neoplasms (MPN) were equally sensitive against pazopanib and imatinib, two other such point mutations showed differential sensitivity, with Y849S being more sensitive to imatinib and N659S being more sensitive to pazopanib. When evaluating the effect of pazopanib on acute myeloid leukemia cell lines, we found that imatinib inhibited the cell growth and reduced colony forming units of Kasumi-1 and BCR-ABL positive K562 but not MV4-11 or NB-4 cells, while Kasumi-1, MV4-11, and NB-4 cells were sensitive towards pazopanib treatment. Moreover, while the clonogenic growth of bone marrow-derived cells from two control patients (lymphoma or AML in remission) were only reduced to 75% of control with pazopanib treatment in vitro (100 nM), 100 nM pazopanib decreased colony growth to 18% in another patient with AML at diagnosis. Conclusion:Our data suggest that pazopanib may be active against a variety of myeloid neoplasms and that clinical studies assessing its efficacy are warranted. Also, cells may show differential sensitivity against the PDGFR and KIT inhibitors pazopanib and imatinib. Disclosures:Koschmieder:GlaxoSmithKline: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding. Off Label Use: Data on in vitro activity of pazopanib and imatinib in MPN and AML discussed. Krug:MedA Pharma: Honoraria; Novartis: Honoraria; Alexion: Honoraria; Boehringer Ingelheim: Research Funding; Sunesis: Honoraria. Müller-Tidow:Novartis: Research Funding.

Mer Receptor Tyrosine Kinase Is Over-Expressed In and Contributes to Oncogenesis In Acute Myeloid Leukemia

Abstract 1390The abnormal activation of tyrosine kinases in pediatric leukemias has been associated with a poor prognosis, and provides a potential focus for targeted therapy. Pediatric acute myelogenous leukemia (AML) is known to be particularly difficult to treat successfully. The development of therapy for AML targeted against a specific cancer-promoting signaling pathway would potentially allow for a more efficacious clinical response with less therapy-associated toxicity. The Mer Tyrosine Kinase (TK), a transmembrane receptor in the TAM family, is known to regulate intracellular pathways promoting cell survival and proliferation in a number of malignancies, but has not previously been explored in AML.We assessed the prevalence of Mer TK expression in AML. Western blot and flow cytometric analysis demonstrated aberrant expression of Mer TK in 80% (13 of 15) of AML cell lines. Similarly, greater than 85% (24 of 28) of samples from newly diagnosed pediatric AML patients expressed Mer TK on leukemic blasts. In addition, 5 of 6 pediatric patients with relapsed or refractory AML had increased or equivalent Mer expression by flow cytometry relative to diagnostic samples.To assess whether Mer plays a role in proliferation in AML, we investigated downstream signaling pathways in the Nomo-1 and Kasumi-1 AML cell lines. Phosphoarray and western blot analysis demonstrated increased phospho-Erk 1/2, phospho-Akt, phospho-mTOR and phospho-MSK1 following treatment with Gas6, the Mer ligand. These data demonstrate activation of pathways which are known to aid in malignant cell survival.To assess the effect of Mer TK inhibition on myeloblast phenotype, we used two different shRNA constructs to decrease expression of Mer by >50% in the Nomo-1 and Kasumi-1 cell lines. The ability of these cell lines to evade apoptosis was determined by flow cytometry following staining with propidium iodide and Yo-Pro-1-iodide. Compared to wild-type Nomo-1 and Kasumi-1, the cell lines expressing decreased levels of Mer demonstrated two to four times more apoptosis in response to serum starvation (p<0.5). Additionally, myeloblast proliferative capacity was assessed using methylcellulose colony forming assays. Compared to wild-type, the AML cell lines expressing reduced levels of Mer demonstrated a 40–70% decrease in total colony forming units (p<0.5).To explore how knockdown of Mer affects myeloblast survival in vivo, we used a mouse xenograft model. Sub-lethally irradiated NSG mice were injected intravenously with wild-type Nomo-1 or Mer knock-down Nomo-1 lines and tumor-free survival was determined. Kaplan-Meier curves were generated and demonstrated a statistically significant difference in survival between mice injected with wild-type Nomo-1 cells and those injected with a Nomo-1 Mer knock-down cell line (20 versus 43 days, p<0.1). These data demonstrate a role for Mer in acute myelogenous leukemogenesis in vivo and suggest that inhibition of Mer TK may have a clinically significant effect in patients as a targeted therapy in the treatment of human AML. Disclosures:No relevant conflicts of interest to declare.

PIG7, transactivated by AML1, promotes apoptosis and differentiation of leukemia cells with AML1–ETO fusion gene

AML1, the potent transcription factor in hematopoiesis, is antagonized by AML1-ETO in t(8;21) leukemia. Our previous study showed that the differentiation and apoptosis of Kasumi-1 induced by sodium phenylbutyrate (PB), were accompanied by significant upregulation of PIG7 and AML1b (one of the AML1 isoforms). Here, we further investigated the relationship between AML1b and PIG7, also the effects of PIG7 on leukemia cells. The results demonstrated that exogenous AML1b could upregulate PIG7 expression in HEK-293 and CV-1 cells in sequence-specific and dosage-dependent manners, and this effect was antagonized by AML1-ETO. The specific AML1-binding site required for p53-induced gene 7 (PIG7) transactivation was located between nucleotides -1511 and -1503 in the PIG7 promoter. Overexpression of PIG7 could induce the apoptosis and differentiation of Kasumi-1 and SKNO-1 cells, but showed less effect on NB4 cells directly. Moreover, ectopic expression of PIG7 could sensitize these cell lines to PB or all-trans retinoic acid, respectively, which could then be abrogated by downregulation of PIG7 expression. Furthermore, the primary acute myeloid leukemia cells showed similar response to the ectopic expression of PIG7. In conclusion, PIG7 could be transactivated by AML1, which subsequently induces differentiation and apoptosis of leukemia cells, especially those with AML1-ETO fusion gene.

Shear Stress Regulates Adhesion and Rolling of CD44+ Leukemic and Hematopoietic Progenitor Cells on Hyaluronan

Leukemic cells and human hematopoietic progenitor cells expressing CD44 receptors have the ability to attach and roll on hyaluronan. We investigated quantitatively the adhesion behavior of leukemic cell lines and hematopoietic progenitor cells on thin films of the polysaccharides hyaluronan and alginate in a microfluidic system. An applied flow enhances the interaction between CD44-positive cells and hyaluronan if a threshold shear stress of 0.2 dyn/cm 2 is exceeded. At shear stress ∼1 dyn/cm 2, the cell rolling speed reaches a maximum of 15 μm/s. Leukemic Jurkat and Kasumi-1 cells lacking CD44-expression showed no adhesion or rolling on the polysaccharides whereas the CD44-expressing leukemic cells KG-1a, HL-60, K-562, and hematopoietic progenitor cells attached and rolled on hyaluronan. Interestingly, the observations of flow-induced cell rolling are related to those found in the recruitment of leukocytes to inflammatory sites and the mechanisms of stem-cell homing into the bone marrow.

Heat shock protein 90 inhibition results in altered downstream signaling of mutant KIT and exerts synergistic effects on Kasumi-1 cells when combining with histone deacetylase inhibitor

KIT mutations may be associated with a poor prognosis in t(8;21) AML. Heat shock protein 90 (Hsp90) is a molecular chaperone frequently used by cancer cells to stabilize mutant oncoproteins. Inhibition of Hsp90 by 17-allylamino-17-demethoxygeldanamycin (17-AAG) disrupted downstream signaling pathways of mutant KIT in Kasumi-1 cells. AML1-ETO fusion gene and mutated KIT act as “two-hit” factors in Kasumi-1 cells. Histone deacetylation (HDAC) inhibitors sodium phenylbutyrate (PB) and valproic acid (VPA) block AML1-ETO. Co-treatment with 17-AAG and PB or 17-AAG and VPA resulted in a synergistic effect in Kasumi-1 cells. Our results confirmed that Hsp90 and mutated KIT were valid molecular targets in the therapy of AML.

Curcumin reduces expression of Bcl-2, leading to apoptosis in daunorubicin-insensitive CD34+ acute myeloid leukemia cell lines and primary sorted CD34+ acute myeloid leukemia cells

BackgroundAcute myeloid leukemia (AML) is an immunophenotypically heterogenous malignant disease, in which CD34 positivity is associated with poor prognosis. CD34+ AML cells are 10-15-fold more resistant to daunorubicin (DNR) than CD34- AML cells. Curcumin is a major component of turmeric that has shown cytotoxic activity in multiple cancers; however, its anti-cancer activity has not been well studied in DNR-insensitive CD34+ AML cells. The aim of this study was to therefore to explore curcumin-induced cytotoxicity in DNR-insensitive CD34+ AML cell lines (KG1a, Kasumi-1), DNR-sensitive U937 AML cells, and primary CD34+ AML bone-marrow-derived cells.MethodsPrimary human CD34+ cells were isolated from peripheral blood mononuclear cells or bone marrow mononuclear cells using a CD34 MicroBead kit. The growth inhibitory effects of curcumin were evaluated by MTT and colony-formation assays. Cell cycle distribution was examined by propidium iodide (PI) assay. Apoptosis was analyzed by Wright-Giemsa, Hoechst 33342 and Annexin-V/PI staining assays. The change in mitochondrial membrane potential (MMP) was examined by JC-1 staining and flow cytometry. Expression of apoptosis-related proteins was determined by reverse transcription-polymerase chain reaction and Western blotting. Short interfering RNA (siRNA) against Bcl-2 was used in CD34+ KG1a and Kasumi-1 cells incubated with/without DNR.ResultsCurcumin inhibited proliferation and induced apoptosis and G1/S arrest in both DNR-insensitive KG1a, Kasumi-1 and DNR-sensitive U937 cells. Curcumin-induced apoptosis was associated with reduced expression of both Bcl-2 mRNA and protein, subsequent loss of MMP, and activation of caspase-3 followed by PARP degradation. Curcumin synergistically enhanced the cytotoxic effect of DNR in DNR-insensitive KG1a and Kasumi-1 cells, consistent with decreased Bcl-2 expression. Accordingly, siRNA against Bcl-2 increased the susceptibility of KG1a and Kasumi-1 cells to DNR-induced apoptosis. More importantly, curcumin suppressed Bcl-2 expression, selectively inhibited proliferation and synergistically enhanced the cytotoxicity of DNR in primary CD34+ AML cells, while showing limited lethality in normal CD34+ hematopoietic progenitors.ConclusionCurcumin down-regulates Bcl-2 and induces apoptosis in DNR-insensitive CD34+ AML cell lines and primary CD34+ AML cells.

The HDAC class I-specific inhibitor entinostat (MS-275) effectively relieves epigenetic silencing of the LAT2 gene mediated by AML1/ETO

The chromosomal translocation (8;21) fuses the hematopoietic transcription factor AML1 (RUNX1) with ETO (RUNX1T1, MTG8), resulting in the leukemia-specific chimeric protein AML1/ETO. This fusion protein has been implicated in epigenetic silencing, recruiting histone deacetylases (HDACs) and DNA methyltransferases to target promoters. Previously, we have identified a novel in vivo AML1/ETO target gene, LAT2 (NTAL/LAB/WBSCR5), which is involved in FcɛR I, c-Kit, B-cell and T-cell receptor signalling. We have now addressed the molecular mechanisms of AML1/ETO-mediated LAT2 repression. In Kasumi-1 cells, where AML1/ETO bound to the LAT2 gene, small interfering RNA (siRNA)-mediated AML1/ETO depletion caused upregulation of LAT2, suggesting a possible direct mechanism of repression. Expression of AML1/ETO was associated with a decrease in acetylation of histones H3, H3K9 and H4, and an increase in H3K9 and H3K27 trimethylation. The class I-specific HDAC inhibitors entinostat (MS-275) and mocetinostat (MGCD0103) induced LAT2 expression specifically in AML1/ETO-expressing cells, resulting in induction of several activating histone marks on the LAT2 gene, including trimethylation of histone H3K4. The combination of entinostat and decitabine increased acetylation of histones H3 and H4, as well as LAT2 mRNA expression, in an at least additive fashion. In conclusion, several repressive histone modifications mark the LAT2 gene in the presence of AML1/ETO, and LAT2 gene derepression is achieved by pharmacological inhibition of HDACs.

Cysteine Proteases in acute myeloid leukaemia

We have previously identified calpains as important modulators of AML1/MTG8-dependent leukaemogenesis. Like caspases and cathepsins, calpains belong to the family of cysteine proteases. By application of cysteine protease inhibitors and siRNAs we investigated individual cysteine proteases essential for leukaemic growth, maintenance and progression. Cells were transfected with siRNAs using electroporation; knockdown was confirmed by real-time RT-PCR. Cysteine protease inhibitors were applied in various concentrations. Proliferation was determined by cytotoxicity assays and cell cycle analysis. Clonogenicity was assessed in semisolid medium. Application of four different inhibitors targeting calpains and cathepsins impinges on proliferation and clonogenicity of t(8;21)-positive Kasumi-1 cells. Furthermore, these inhibitors also show cytotoxicity in other t(8;21)-positive and -negative leukaemia cell lines. Calpain and cathepsin inhibition has anti-leukaemic effects and is not restricted to AML1/MTG8-positive AML. Considering specificity of the inhibitors, the data suggests that – beside the calpains – cathepsin B and L are important therapeutic targets in leukaemia.