Cell Lines KG-1 Research Articles

Antagonizing Retinoic Acid Receptors Increases Myeloid Cell Production by Cultured Human Hematopoietic Stem Cells

Activities of the retinoic acid receptor (RAR)α and RARγ are important to hematopoiesis. Here, we have investigated the effects of receptor selective agonists and antagonists on the primitive human hematopoietic cell lines KG1 and NB-4 and purified normal human hematopoietic stem cells (HSCs). Agonizing RARα (by AGN195183) was effective in driving neutrophil differentiation of NB-4 cells and this agonist synergized with a low amount (10 nM) of 1α,25-dihydroxyvitamin D3 to drive monocyte differentiation of NB-4 and KG1 cells. Treatment of cultures of human HSCs (supplemented with stem cell factor ± interleukin 3) with an antagonist of all RARs (AGN194310) or of RARα (AGN196996) prolonged the lifespan of cultures, up to 55 days, and increased the production of neutrophils and monocytes. Slowing down of cell differentiation was not observed, and instead, hematopoietic stem and progenitor cells had expanded in number. Antagonism of RARγ (by AGN205728) did not affect cultures of HSCs. Studies of CV-1 and LNCaP cells transfected with RAR expression vectors and a reporter vector revealed that RARγ and RARβ are activated by sub-nM all-trans retinoic acid (EC50–0.3 nM): ~50-fold more is required for activation of RARα (EC50–16 nM). These findings further support the notion that the balance of expression and activity of RARα and RARγ are important to hematopoietic stem and progenitor cell expansion and differentiation.

Kefir induces apoptosis and inhibits cell proliferation in human acute erythroleukemia.

Acute erythroleukemia is an uncommon subtype of acute myeloid leukemia which has been considered to be a subtype of AML with a worse prognosis. Intensive chemotherapy is the first line of treatment. In recent years, the effect of kefir on some malignancies has been experimented. Kefir is a kind of beverage, which obtained by incubation of kefir grains with raw milk. Kefir grains are a symbiotic complex of different kinds of yeasts and bacteria, especially lactic acid bacteria which gather in a mostly carbohydrate matrix, named kefiran. We investigated the effect of kefir on acute erythroleukemia cell line (KG-1) and peripheral blood mononuclear cells (PBMCs). The cell line and PBMCs were treated with different doses of kefir and milk and incubated for three different times. We used Polymixin B to block the lipopolysaccharide and NaOH (1mol/l) to neutralize the acidic media. Viability was detected by MTT assay. Apoptosis and necrosis were assessed by annexin-propidium iodide staining. Our results showed that kefir induced apoptosis and necrosis in KG-1 cell line. It was revealed that kefir decreased proliferation in erythroleukemia cell line. We did not observe a remarkable effect of kefir on PBMCs. Our study suggested that kefir may have potential to be an effective treatment for erythroleukemia.

TET2 Downregulation Leads to AML Cells Sensitive to Decitabine

Background: Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults and have very lethal rate. Chemotherapy is the main method to treat AML, but the complete remission rate is still not very optimal. With the development of genetic and molecular biology technologies, more and more molecular biomarkers are found, some of them are useful for us to evaluate the prognosis and can help us to tailor the treatment plan for different patients. TET2, a member of the ten-eleven-translocation(TET) family genes which can modify DNA by catalyzing the conversion of 5mehtyl-cytosine(5-mC) to 5-hydroxymethyl-cytosine(5-hmC), is often inactivated through loss-of-function mutation and deletion in myeloid malignancies. Recent clinical research reported that the lower the expression of TET2 in MDS and AML patients, the better the response to decitabine (DAC, a demethylation agent) will be. However, the mechanism of the phenomenon is still unknown. Our investigation is trying to uncover the mechanism how TET2 protein levels are negatively related with AML sensitivity to decitabine.Methods: We detected TET2 mRNA expression level in acute leukemia cell lines, bone marrow AML specimens and peripheral blood mononuclear cells from healthy donors by semiquantitative real time polymerase chain reaction (qRT-PCR). Western blot is also applied to detect TET2 protein expression. In order to access TET2 methylation status, we used the methylation-specific PCR. And we also checked the mutant status of TET2 in U937 and KG-1 cell line. CCK8 and flow cytometry are used to detect cell proliferation rate, cell apoptosis, and cell cycle profile. Also, we developed TET2 knock-down and overexpression lentivirus to transfect AML cell lines to explore the mechanism why TET2 expression level is related to the response of DAC. Last, gene array is used to compare gene expression level changes between TET2 knock-down cell lines (or TET2 overexpression cell lines) and the control cell lines.Results: The AML cell lines (KG-1, U937, Kasumi, HL-60, THP-1) and AML patients specimens express lower TET2 than that of PBMC from the healthy donor (P<0.05). Among AML cell lines, U937 barely expresses TET2, while KG-1 expresses TET2 relatively higher than other AML cell lines. The methylation-specific PCR showed that TET2 in U937 was partially methylated while KG-1 was not. After using decitabine to treat U937 cell line, the TET2 methylation status was attenuated. And all the exons of TET2 were not detected any mutation in KG-1 AND U937. Then, we used CCK8 to compare the response difference to DAC between U937 and KG-1 and found that U937 is much more sensitive to DAC rather than KG-1 (P<0.05). Next, we constructed a TET2 shRNA to transfect KG-1, both qRT-PCR and WB were used to verification the knock-down efficiency. Again, CCK8 told us that KG-1 TET2 knock-down cells was more sensitive to DAC than KG-1 NC cells. Flow cytometry identified that cell cycle profile were altered between KG-1 TET2 knock-down cells and KG-1 NC cells. Gene array (KG-1 TET2 KD and KG-1 NC) showed that the expression levels of cell cycle related genes (e.g. CCNB2,RBL1), DNA replication related genes (e.g. PRIM1, RCF3, FEN1) and many other function genes were changed between the knock-down and control cell line.Conclusion: Our study showed that the sensitivity to decitabine of AML cell lines is related to TET2 expression level, knock-down TET2 in KG-1 can increase its vulnerability to decitbine. And the mechanism may be related to the changing expression levels of the genes which regulating cell cycles and DNA replication. DisclosuresNo relevant conflicts of interest to declare.

SGN-CD123A, a Pyrrolobenzodiazepine Dimer Linked Anti-CD123 Antibody Drug Conjugate, Demonstrates Effective Anti-Leukemic Activity in Multiple Preclinical Models of AML

Survival expectations for acute myeloid leukemia (AML) patients remain poor, highlighting the need for further treatment options. The majority of AML blasts express CD123, the alpha subunit of the IL-3 receptor, which regulates the proliferation, survival and differentiation of hematopoietic cells. CD123 is also robustly expressed on leukemic stem cells and is a marker for minimal residual disease (MRD, Roug et al. 2012). Poor prognosis has previously been associated with elevated expression of CD123 on leukemic stem cells and blasts (Vergez et al. 2011, Testa et al. 2002). These findings identify CD123 as a rational therapeutic target in AML.Here we report the preclinical development of a novel CD123-directed immunoconjugate SGN-CD123A, consisting of a humanized anti-CD123 antibody conjugated to a highly potent DNA binding pyrrolobenzodiazepine (PBD) dimer drug via a protease-cleavable dipeptide linker. An engineered cysteine on each heavy chain attaching the PBD dimer to the antibody allows uniform drug loading of approximately two PBD dimers per antibody. Fluorescence microscopy studies show that SGN-CD123A is rapidly internalized and traffics to lysosomes within hours of binding to CD123-positive AML cells. Uptake of the antibody-drug-conjugate (ADC) induced DNA damage as measured by dose-and time-dependent increases in the phosphorylation of histone 2AX (γH2AX) and cell death associated with G2-M cell cycle arrest, caspase-3 activity, formation of cleaved poly ADP-ribose polymerase, and DNA fragmentation in target cells.The anti-leukemic activity of SGN-CD123A was assessed in cytotoxicity assays in 12 AML cell lines and 23 primary AML patient samples with variable cytogenetic abnormalities (favorable, intermediate and adverse) and multi-drug resistance (MDR) status. SGN-CD123A was highly active in 11 of 12 AML cell lines tested (mean IC50, 6 ng/ml; range of 0.02 to 38 ng/ml), including 4 of 5 MDR-positive cell lines, whereas it was inactive in CD123-negative HEL92.1.7 AML cells. SGN-CD123A was also active against 20 of 23 primary samples isolated from AML patients (mean IC50 of responsive samples, 0.8 ng/mL; range of 0.06 to 2.5 ng/ml). In both AML panels, molecular abnormalities, including the presence of a p53 mutation, FLT3-ITD, as well as MDR positivity, did not affect the in vitro cytotoxic activity of SGN-CD123A.In vivo antitumor activity was evaluated in AML xenograft models established with CD123-positive, MDR-negative Molm-13, HNT-34, and THP-1 cell lines and the MDR-positive KG-1 cell line. In all of the in vivo models, a single dose of SGN-CD123A delivered significant antitumor activity. SGN-CD123A dosed once at 10 mcg/kg yielded complete cures and significant survival advantage in the Molm-13 disseminated model of AML (p < 0.0001 compared to untreated or control ADC groups). Durable complete regressions were observed with a single dose of 25 or 75 mcg/kg in the MDR-negative HNT-34 subcutaneous model (p =0.0019 to control ADC group). In the THP-1 model, a single 100 mcg/kg dose of SGN-CD123A yielded durable complete regressions in 2 of 8 mice (p=0.0003 to untreated) whereas a higher dose of 300 mcg/kg gave complete tumor regressions in all mice (p < 0.0001 to untreated group). SGN-CD123A was also effective in a MDR-positive model of AML. A single dose of 100 mcg/kg SGN-CD123A significantly decreased tumor growth (p=0.003 to controls) whereas a single dose of 300 mcg/kg yielded durable complete regressions compared to the control groups in the KG-1 subcutaneous model of MDR-positive AML (p =0.008). Early evidence of the antitumor activity of SGN-CD123A was found in tumors harvested from THP-1 mice. Within 48h of dosing with SGN-CD123A, tumor cells showed elevated levels of the DNA damage marker γH2AX and changes in nuclear morphology.These data demonstrate that SGN-CD123A exhibits significant antitumor activity against a broad panel of primary AML samples and in preclinical models of MDR-positive AML that are characteristically resistant to chemotherapy. CD123-directed delivery of PBD may represent a promising new approach for the treatment of AML. DisclosuresSutherland:Seattle Genetics, Inc.: Employment. Yu:Seattle Genetics, Inc: Employment, Equity Ownership. Walter:Seattle Genetics, Inc: Consultancy, Research Funding. Westendorf:Seattle Genetics, Inc: Employment. Valliere-Douglass:Seattle Genetics, Inc: Employment. Pan:Seattle Genetics, Inc: Employment. Sussman:Seattle Genetics, Inc: Employment. Anderson:Seattle Genetics, Inc: Employment. Zeng:Seattle Genetics, Inc: Employment. Stone:Seattle Genetics, Inc: Employment. Klussman:Seattle Genetics, Inc: Employment. Ulrich:Seattle Genetics, Inc: Employment. Jonas:Seattle Genetics, Inc: Employment. Senter:Seattle Genetics, Inc: Employment. Drachman:Seattle Genetics, Inc: Employment. Benjamin:Seattle Genetics, Inc: Employment.

MiR-181a modulates acute myeloid leukemia susceptibility to natural killer cells

Although daunorubicin (DNR) is the most widely used anthracycline to treat acute myeloid leukemia (AML), resistance to this drug remains a critical problem. The aim of this study was to investigate the relationship between AML resistance to daunorubicin and susceptibility to natural killer (NK) cell-mediated cell lysis, and the putative expression of miRs. For this purpose, we used the parental AML cell lines U-937 and KG-1 and their equivalent resistant U937(R) and KG-1(R) cell lines. We demonstrate for the first time that the acquisition of resistance to DNR by the parental cell lines resulted in the acquisition of cross-resistance to NK cell-mediated cytotoxicity. miR microarray analysis revealed that this cross-resistance was associated with miR-181a downregulation and the subsequent regulation of MAP3K10 and MAP2K1 tyrosine kinases and the BCL-2 (BCL-2 and MCL-1) family. Overexpression of miR-181a in AML blasts resulted in the attenuation of their resistance to DNR and to NK-cell-mediated killing. These data point to a determinant role of miR-181a in the sensitization of leukemic resistant cells to DNR and NK cells and suggest that miR-181a may provide a promising option for the treatment of immuno- and chemo-resistant blasts.

Can the KG1 cell line be used as a model of dendritic cells and discriminate the sensitising potential of chemicals?

The KG1 myeloid leukaemia was used as source of dendritic cells (DC) to discriminate between respiratory and contact sensitising chemicals. A cocktail of cytokines was used to differentiate KG1 to dendritic like cells (termed dKG1) and the effects of nine chemicals (respiratory and contact sensitisers) and an irritant control on surface marker expression, 'antigen presenting' function and cytokine expression investigated. The stability of these chemicals when dissolved was characterised using MALDI ToF MS. A Hill plot model was used with the cellular viability data to quantify the lethal dose 50% (LD50) and a maximum sub toxic concentration of each chemical defined. Cytokine expression by the treated dKG1 was quantified using multiplex immunobead analysis. Whilst dKG1 cells were morphologically similar to DCs, expression of specific surface markers was not typical for DCs derived from healthy precursor cells. When the chemicals were applied at defined sub toxic doses no effects on dKG1 phenotype, function, or cytokine expression, attributable to the sensitisation properties were discriminated. However, dKG1 cells were much more sensitive to the toxic effects of these chemicals compared to the parent KG1 cells. Only 4 of the 9 chemicals tested were stable when dissolved indicating that the effect of sensitising chemicals on antigen presenting cells may be related to species other than the parent compound.

Abstract 2462: In Vivo efficacy of a novel anti-CD33 targeted thorium conjugate (TTC) in mouse models of acute myeloid leukemia (AML)

Abstract Introduction: The sialoadhesin receptor CD33 is a well-established target in the treatment of acute myeloid leukemia (AML). CD33 is expressed on leukemic blasts in AML and possibly also on leukemic stem cells in some patients (1). Thorium-227, an alpha-particle emitting radionuclide with a half-life of 18.7 days, is being studied for use as a possible cytotoxic agent. We have developed a novel Targeted Thorium Conjugate (TTC), comprising an anti-CD33 monoclonal antibody conjugated to a chelator for the efficient delivery of high-energy alpha-particles. We present data from in vitro cell cytotoxicity assays as well as biodistribution and efficacy data from preclinical animal models of AML. Experimental procedures: Antibody conjugates were prepared and labeled with 227Th at room temperature giving high radiochemical yields and purity. In vitro cell cytotoxicity experiments were carried out on HL-60 cells and the multi-drug resistant cell line KG-1 by measuring cell viability and monitoring apoptosis. In vivo efficacy and biodistribution were performed in a sub-cutaneous model of AML in athymic nude (nu/nu) mice using HL-60 cells. The 227Th-anti-CD33 conjugate was further tested in a disseminated model of AML using HL-60 cells, injected intravenously 5 days prior to treatment in CB.17 SCID mice with hind-leg paralysis as endpoint. Results: The 227Th-anti-CD33 conjugate was a potent and specific inducer of in vitro cell cytotoxicity independent of MDR-status. Mechanistic studies demonstrated cell arrest in G2 phase with cytotoxicity associated with apoptotic pathways. In vivo biodistribution in a sub-cutaneous model demonstrated a high tumor to blood ratio (TBR ∼ 6) at day 7 post injection based on measured 227Th-activity. Significant efficacy was achieved in two models of AML using human HL-60 cells. In athymic nude mice, a single injection of 227Th-anti-CD33 conjugate at 700 kBq/kg resulted in complete regression of established sub-cutaneous tumors. Importantly, no significant loss in body weight was observed during the course of the study, demonstrating that the dose was well tolerated. In parallel, statistically significant dose dependency of 227Th-anti-CD33 conjugate was demonstrated in a disseminated model of AML with median survival times (MST) of 90 days and 116 days for animals receiving 50 and 150 kBq/kg. Around 80% of animals dosed with either 2 × 150 or 1 × 300 kBq/kg where still alive at the end of the study with no gross pathological findings observed. Conclusions: The data presented are supportive of the further investigation of the 227Th-anti-CD33 conjugate for the treatment of AML.

Endothelial cell derived angiocrine support of acute myeloid leukemia targeted by receptor tyrosine kinase inhibition

In acute myeloid leukemia (AML), refractory disease is a major challenge and the leukemia microenvironment may harbor refractory disease. Human AML cell lines KG-1 and HL-60 expressed receptors also found on endothelial cells (ECs) such as VEGFRs, PDGFRs, and cKit. When human AML cells were co-cultured with human umbilical vein endothelial cells (HUVECs) and primary bone marrow endothelial cell (BMECs), the AML cells were more resistant to cytarabine chemotherapy, even in transwell co-culture suggesting angiocrine regulation. Primary BMECs secreted significantly increased levels of VEGF-A and PDGF-AB after exposure to cytarabine. Pazopanib, a receptor tyrosine kinase inhibitor (RTKI) of VEGFRs, PDGFRs, and cKit, removed EC protection of AML cells and enhanced AML cell sensitivity to cytarabine. Xenograft modeling showed significant regression of AML cells and abrogation of BM hypervascularity in RTKI treated cohorts. Together, these results show direct cytotoxicity of RTKIs on AML cells and reversal of EC protection. Combining RTKIs with chemotherapy may serve as promising therapeutic strategy for patients with AML.

New benzimidazole-derived isothioureas as potential antileukemic agents--studies in vitro.

Isothioureas are a class of amphiphilic compounds carrying a highly basic isothiourea group of pKa ranging between 10 and 11. Hence, they exist in protonated (cation) form at physiological pH, a characteristic is of key importance for their pharmacological activity. Recently, we have found that a number of S-pentabromobenzylisothiourea derivatives show substantial cytotoxicity toward a variety of human glioblastoma, leukemia, and adenocarcinoma cell lines. Whereas there is a growing body of data on aliphatic and alkylaromatic isothioureas, little attention was given to this day to heterocyclic isotiourea derivatives. Here we report on the synthesis and pharmacological in vitro properties of 10 novel S-(benzimidazol-2-ylmethyl)- and S-(5,6-dichlorobenzimidazol-2-ylmethyl)isothiourea derivatives. The compounds were obtained by the condensation of the respective 2-chloromethyl benzimidazoles with various substituted N(N')-thioureas. Besides the essential physicochemical characteristics (H-NMR, UV, elemental analysis) of the new compounds, their log P values, which are of key importance for in vivo drug distribution and interactions, were determined. Pharmacological (anticancer) activity of the compounds was evaluated based on their ability to induce apoptosis in exponentially growing cultures of the human acute myelogenous leukemia cell line KG-1; the apoptosis was assessed with a variety of flow cytometric methods. Of the novel compounds tested, the most potent apoptosis inducer in KG-1 cells was N-methyl-S- (5,6-dichloro-1H-benzimidazol-2-ylmethyl)isothiouronium chloride (ClBMMe).

Consequences of combining siRNA-mediated DNA methyltransferase 1 depletion with 5-aza-2'-deoxycytidine in human leukemic KG1 cells.

5-azacytidine and 5-aza-2'-deoxycytidine are clinically used to treat patients with blood neoplasia. Their antileukemic property is mediated by the trapping and the subsequent degradation of a family of proteins, the DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B) leading to DNA demethylation, tumor suppressor gene re-expression and DNA damage. Here we studied the respective role of each DNMT in the human leukemia KG1 cell line using a RNA interference approach. In addition we addressed the role of DNA damage formation in DNA demethylation by 5-aza-2'-deoxycytidine. Our data show that DNMT1 is the main DNMT involved in DNA methylation maintenance in KG1 cells and in mediating DNA damage formation upon exposure to 5-aza-2'-deoxycytidine. Moreover, KG1 cells express the DNMT1 protein at a level above the one required to ensure DNA methylation maintenance, and we identified a threshold for DNMT1 depletion that needs to be exceeded to achieve DNA demethylation. Most interestingly, by combining DNMT1 siRNA and treatment with low dose of 5-aza-2'-deoxycytidine, it is possible to uncouple DNA damage formation from DNA demethylation. This work strongly suggests that a direct pharmacological inhibition of DNMT1, unlike the use of 5-aza-2'-deoxycytidine, should lead to tumor suppressor gene hypomethylation and re-expression without inducing major DNA damage in leukemia.

P8.04 - Comparison of Human Nucleoside Transporter Interactions with BCR-ABL Kinase Inhibitors

ABSTRACT Introduction: Human nucleoside transporters (hNTs) regulate cellular influx and disposition of anticancer nucleoside drugs. Cytarabine and fludarabine uptake in leukemic cells, which occurs primarily via human nucleoside transporter 1 (hENT1), was shown to be inhibited by imatinib and nilotinib [ 1 , 2 ]. We studied interactions of the BCR-ABL kinase inhibitors bosutinib, dasatinib, imatinib, nilotinib and ponatinib with five human nucleoside transporters (hENT1-2 and hCNT1-3) that mediate cellular uptake of various anticancer nucleoside drugs. Materials and methods: hNT interactions with bosutinib, dasatinib, imatinib, nilotinib and ponatinib were assessed by comparing their relative abilities to inhibit [3H]uridine uptake in yeast producing each of the recombinant hNTs individually. Inhibition of [3H]uridine, [3H]cytarabine, [3H]fludarabine and [3H]cladribine uptake and long-term accumulation was examined in the CEM cell line. Inhibition of [3H]uridine uptake was also studied in the K562 and KG1 cell lines. Results: BCR-ABL TKIs inhibited recombinant yeast hENT1 and hENT2 with some inhibiting more than others. One or more hCNTs was inhibited by bosutinib, ponatinib, dasatinib and imatinib whereas nilotinib had no effect. In CEM cells (possess only hENT1) nilotinib inhibited hENT1 most potently followed by bosutinib, ponatinib, dasatinib and imatinib with IC50 values of ± 0.7, 2.0, 3.0, 8 and 16 µM, respectively. Accumulation of [3H]cytarabine, cladribine and fludarabine in CEM cells was inhibited by all five BCR-ABL TKIs. Experiments are underway to figure out mechanisms of hNT inhibition by these TKIs. Conclusions: All five BCR-ABL TKIs inhibited hENT1 and hENT2 to different extent leading to reduced intracellular nucleoside accumulation. In addition at least one or more hCNTs were inhibited except by nilotinib. acknowledgements: This research was funded by the Alberta Cancer Board Research Initiative Program (M.B.S., C.E.C), and the Canadian Cancer Society Research Institute (C.E.C.).

Hematopoietic cancer cell lines can support replication of Sabin poliovirus type 1.

Viral vaccines can be produced in adherent or in suspension cells. The objective of this work was to screen human suspension cell lines for the capacity to support viral replication. As the first step, it was investigated whether poliovirus can replicate in such cell lines. Sabin poliovirus type 1 was serially passaged on five human cell lines, HL60, K562, KG1, THP-1, and U937. Sabin type 1 was capable of efficiently replicating in three cell lines (K562, KG1, and U937), yielding high viral titers after replication. Expression of CD155, the poliovirus receptor, did not explain susceptibility to replication, since all cell lines expressed CD155. Furthermore, we showed that passaged virus replicated more efficiently than parental virus in KG1 cells, yielding higher virus titers in the supernatant early after infection. Infection of cell lines at an MOI of 0.01 resulted in high viral titers in the supernatant at day 4. Infection of K562 with passaged Sabin type 1 in a bioreactor system yielded high viral titers in the supernatant. Altogether, these data suggest that K562, KG1, and U937 cell lines are useful for propagation of poliovirus.

MDR1 Mediated Drug Resistance to a Histone Methyltansferase Inhibitor (KMT)

Aberrant histone methylation is emerging as a major contributor to oncogenesis, and inhibitors of histone lysine methyltransferases (KMTs) are starting to enter early clinical studies. Currently, inhibitors for two KMTs are in phase I/II clinical trials, an inhibitor of the histone 3 lysine 79 (H3K79) methyltransferase DOT1L for MLL-rearrange leukemias (NCT02141828), and inhibitors for the H3K27 methyltransferase EZH2 for lymphomas or solid tumors with activating EZH2 mutations (NCT01897571 and NCT02082977). The next few months and years are likely to see more KMT inhibitors entering clinical development, such as compounds directed against the H3K36 methyltransferase MMSET/NSD2/WHSC1, which is commonly translocated in multiple myeloma and carries activating mutations in a subset of acute lymphoblastic leukemia. Typically, KMT inhibitors are structurally similar and are derived from the methyl donor S-adenosyl-methionine (SAM). Very little is known about possible mechanisms of resistance to these compounds.We exposed a panel of leukemia cell lines to varying concentrations of the DOT1L inhibitor EPZ4777 and assessed the decrease of H3K79 di-methylation by Western Blotting. Our analysis revealed a dose-dependent decrease of H3K79 dimethylation in most cell lines. Interestingly, we identified a pair of cell lines, KG-1 and KG-1a, which were near completely resistant to EPZ4777 mediated DOT1L inhibition, with no effect of 10 μM EPZ4777 (4 day exposure). Even after exposure to EPZ4777 for 15 days, KG-1 showed only a slight decrease in H3K79me2 marking and no decrease in marking was seen for KG-1a up to a concentration of 3 μM. We searched the encode database for expression levels of DOT1L, as well as higher expression of proteins previously described as mediating drug resistance in AML cell lines. We found no significantly higher DOT1L expression level, suggesting that overexpression of the target is not the predominant mechanisms of resistance in KG-1/KG-1a. We confirmed similar baseline H3K79 methylation levels in KG1/KG1a and the other cell lines in our panel by Western Blotting. However, we noted a higher expression of multidrug resistance protein 1 (MDR1/p-glycoprotein/ABCB1) in the KG-1 and KG-1a cell lines compared to the other cell lines in our panel. To explore whether DOT1L inhibition by EPZ4777 could be restored in KG1 and KG1a cells when MDR1 was inhibited, cells were exposed to 0.3 μM EPZ4777 and varying concentrations of Cyclosporine A (CSA), a known inhibitor of MDR1. Increasing concentration of CSA with a fixed dose of 0.3 μM EPZ4777 led to a dose dependent decrease in H3K79me2 in KG-1/KG-1a, suggesting that inhibition of MDR1 increased the intracellularly effective dose of EPZ4777. CSA at a concentration of 0.3 μM and 0.3 μM EPZ4777 near completely abolished H3K79 methylation. In summary, our results suggest that KMT inhibitors can be subject to classic drug resistance mechanisms such as the MDR1/p-glycoprotein/ABCB1 efflux pump. These data provide insight for the use and design of small-molecule inhibitors for KMTs. DisclosuresNo relevant conflicts of interest to declare.

Personalized Drug Combinations for the Treatment of Acute Myeloid Leukemia (AML) Patients

Acute myeloid leukemia (AML) is a highly heterogeneous disorder characterized by the rapid clonal proliferation of blasts derived from hematopoietic progenitor cells, leading to failure of normal hematopoiesis. Although standard therapy, usually including idarubicin and cytarabine, has been used to achieve remission, the long-term survival rates remain low.To show that it is possible to improve existing AML standard therapy, we have used experimental algorithms we have recently developed (iterative algorithms that find effective combinations without a full combinatorial screening) to find selective drug combinations for AML cells. The first search was done using the AML cell line KG-1 for the initial steps and cells from an AML patient for the final step. A normal fibroblast cell line (IMR-90) and normal blood progenitor cells were used as controls for toxicity. Drugs were chosen from those already approved or in clinical trials for AML. As shown in Figure 1, this experimental search was able to achieve selective killing of AML patient cells with a ratio close to 100:1 with respect to non-cancer control cells.To refine the choice of drugs to be used in the search, we recently developed a network model of AML intracellular signal transduction (see www.leukemianetworks.org). The present version (AML 2.1) includes 5667 proteins and 22,218 interactions. The network can be used to integrate genomic and gene expression information from individual patients and to provide a shortlist of drugs to be tested further. Using literature data and our network model we identified three compounds as promising for AML drug combinations: the MEK inhibitor PD0325901, the FLT3 inhibitor Quizartinib and the CDK 4/6 inhibitor Palbociclib. For this experiment, normal progenitor blood cells were the controls. Figure 2 shows the results of 64 experimental tests of the response to different combinations of these drugs. The two AML patients clearly exhibited different drug sensitivities, further supporting the necessity of personalized drug combinations.To improve the clinical relevance of these in vitro studies, we investigated the correlation between clinical and in vitro response to standard AML therapy, using a set of media containing 16 cytokines/growth factors in different combinations. These molecules have been selected using three methods: ligands acting on receptors that are part of gene signatures with prognostic significance in AML; cytokines that promote hematopoietic differentiation of stem cells; and ligands of receptors identified as playing an important role in AML in a recent RNAi screen. During several iterations of our combinatorial search algorithm we studied 185 different media. Figure 3 shows the correlation between clinical response (residual blasts after 28 days of therapy) and response to the same therapy in vitro after 72 h achieved in the last iteration of the search, in a study of six patients. The medium with the highest correlation had a correlation coefficient of 0.99 (p=0.0002). The in vitro response in this medium is clearly able to distinguish patients that respond to standard therapy vs those that do not, as can be seen from the inset of Fig. 3. Some cytokines (BMP-4 and IL-4) were always absent in the group of 14 media with lower correlation shown on the right of Figure 3. The remarkable variation in correlation with clinical response of these media, spanning correlation coefficient values from 0.99 to 0.004, clearly show the importance of optimizing the in vitro microenvironment for prediction of patient drug response.In summary, we identified drug combinations that selectively kill AML primary cells and we optimized media that improve the in vitro prediction of clinical drug response. These methods can together assist in the personalization of AML therapy using patientÕs cells. [Display omitted] [Display omitted] [Display omitted] DisclosuresPiermarocchi:Salgomed Inc.: Equity Ownership. Paternostro:Salgomed Inc.: Equity Ownership.

Lenalidomide Induces Ubiquitination and Degradation of CSNK1A1 in MDS with Del(5q)

▪The immunomodulatory (IMiD) drug lenalidomide is a highly effective treatment for multiple myeloma and myelodysplastic syndrome (MDS) with deletion of chromosome 5q (del(5q)). Recently, we and others demonstrated that lenalidomide activates the CRBN-CRL4 E3 ubiquitin ligase to ubiquitinate IKZF1 and IKZF3. Degradation of these lymphoid transcription factors explains lenalidomide’s growth inhibition of multiple myeloma cells and increased IL-2 release from T cells. However, it is unlikely that degradation of IKZF1 and IKZF3 accounts for lenalidomide’s activity in MDS with del(5q). Instead, we hypothesized that ubiquitination of a distinct CRBN substrate in myeloid cells explains the efficacy of lenalidomide in del(5q) MDS.Applying quantitative proteomics in the myeloid cell line KG-1, we identified a novel target, casein kinase 1A1 (CSNK1A1), that had increased ubiquitination and decreased protein abundance following lenalidomide treatment. CSNK1A1 is encoded in the del(5q) commonly deleted region and is thus a potential lenalidomide target in del(5q) MDS. Previous studies have demonstrated that Csnk1a1 is a therapeutic target in a murine model of acute myeloid leukemia.We validated that lenalidomide treatment decreased CSNK1A1 protein levels in multiple human cell lines in a dose-dependent manner without altering CSNK1A1 mRNA levels. Moreover, lenalidomide treatment increased ubiquitination of CSNK1A1 in cell lines. The decrease in CSNK1A1 protein levels in response to lenalidomide was abrogated by treatment with the proteasome inhibitor MG132 and by Cullin-RING ubiquitin ligase inhibition with MLN4924. CSNK1A1 co-immunoprecipitated with CRBN in the presence of lenalidomide, demonstrating direct interaction of CSNK1A1 with the substrate adaptor for the ubiquitin ligase. Homozygous genetic inactivation of the CRBN gene by CRISPR/Cas9 genome editing in 293T cells eliminated lenalidomide-induced degradation of CSNK1A1. In aggregate, these experiments demonstrate that CSNK1A1 is a CRBN-CRL4 substrate that is ubiquitinated and degraded in the presence of lenalidomide.We next explored how degradation of CSNK1A1 might explain the specificity of lenalidomide for cells with del(5q). ShRNA-mediated knockdown of CSNK1A1 sensitized primary human CD34+ cells to lenalidomide treatment, indicating that haploinsufficiency for CSNK1A1 might increase lenalidomide sensitivity in del(5q) hematopoietic cells. We sought to further validate this finding in a genetically defined Csnk1a1 conditional knockout mouse model. While murine cells are resistant to the effects of IMiDs, murine Ba/F3 cells overexpressing human CRBN (hCRBN), but not murine CRBN, degraded CSNK1A1 in response to lenalidomide. To examine the effect of Csnk1a1 haploinsufficiency on lenalidomide sensitivity, we isolated hematopoietic stem and progenitor cells from Csnk1a1+/- and Csnk1a1+/+ mice and transduced them with a retroviral vector expressing hCRBN. When treated with lenalidmide, Csnk1a1+/- cells expressing hCRBN were depleted over time relative to wild-type controls. The enhanced sensitivity of Csnk1a1+/- cells to lenalidomide was associated with induction of p21 and was rescued by heterozygous deletion of p53, demonstrating a critical downstream role for p53 consistent with clinical observations that TP53 mutations confer lenalidomide resistance.In aggregate, these studies demonstrate that lenalidomide induces the ubiquitination and consequent degradation of CSNK1A1 by the CRBN-CRL4 E3 ubiquitin ligase. del(5q) cells have only one copy of CSNK1A1, so they are selectively depleted over wild-type cells, explaining lenalidomide’s clinical efficacy in del(5q) MDS. Although the idea that heterozygous deletions could be cancer vulnerabilities was first proposed 20 years ago, lenalidomide provides the first example of an FDA-approved and clinically effective drug that derives its therapeutic window from specifically targeting a haploinsufficient gene. DisclosuresEbert:Celgene: Research Funding; Genoptix: Consultancy.

Polymeric nanoparticle-mediated silencing of CD44 receptor in CD34+ acute myeloid leukemia cells

The adhesion receptor CD44 plays an important role in the survival and retention of leukemic stem/progenitor cells (LSPC) within the bone marrow (BM) niche, as well as in the high relapse rates of acute myeloid leukemia (AML). Down-regulating CD44 could be clinically relevant not only for suppression of the deregulated function of LSPC but also in LSPC response to chemotherapeutic agents. Small interfering RNA (siRNA) delivery is a promising approach for AML treatment, and we recently reported effective siRNA delivery into difficult-to-transfect AML cell lines using lipid-substituted polyethylenimine/siRNA complexes (polymeric nanoparticles). In this study, we investigated polymeric nanoparticle-mediated silencing of CD44 in CD34+ LSPC cell models (leukemic KG-1 and KG-1a cell lines) as well as primary AML cells. Polymeric nanoparticle-mediated silencing decreased surface CD44 levels in KG-1, KG-1a and primary AML cells by up to 27%, 30% and 20% at day 3, respectively. Moreover, CD44 silencing resulted in induction of apoptosis in KG-1 cells, reduced adhesion of KG-1 and KG-1a cells to hyaluronic acid-coated cell culture plates and BM-MSC, and decreased adhesion of primary AML cells to BM-MSC. Our results suggest that polymeric nanoparticle-mediated silencing of CD44 might be a useful technique for inhibiting LSPC interactions with their microenvironment, thereby prohibiting leukemia progression or sensitizing LSPC to chemotherapy.

Renin-Angiotensin System (RAS) Expressions in Myeloid Leukemic Cell Lines.

The aim of this study is to search critical renin-angiotensin system (RAS) elements in myeloid leukemic cell lines. Human acute myeloid leukemia (AML) cell lines, KG-1 and HL-60, were cultured. We searched for the gene expression of the major RAS components in KG-1 and HL-60 cell lines by quantitative real-time polymerase chain reaction analysis (qRT-PCR). Angiotensin-converting enzyme I (ACE I) and ACE II mRNA expressions were detected in KG-1 AML cell line. Relative expression of ACE I was higher than ACE II expression in this myeloid leukemic cells. Likewise, RENIN, angiotensinogen (ANGTS), and ACE I mRNA expressions were detected in HL-60 promyelocytic cell line. Relative RENIN expression was the highest, whereas ACE I expression was the lowest in HL60 neoplastic myeloid cells. These findings indicate that there is a biologically active local RAS in the hematopoietic system in normal and pathological states.

Dual PI3K/Akt/mTOR Pathway Inhibitor Potentiate Cytarabine Cytotoxicity For Acute Myeloid Leukemia

Phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signal transduction pathway integrating signals from multiple receptor tyrosine kinases has been firmly established as a major determinant for cell growth, proliferation, and survival in a wide array of solid cancers. PI3K/Akt/mTOR pathway is frequently activated in acute myeloid leukemia (AML) cells and contributes to survival and drug-resistance of AML through various mechanisms. BEZ235 is one of the most promising dual inhibitor of PI3K and mTOR currently under clinical development in solid tumor area. In this study, the potential of BEZ235 was investigated as antileukemic agent using alone or with cytarabinearabinoside (AraC) as combination regimen.AML cell lines KG-1, MV 4-11, THP-1 and HL60 were treated with AraC, BEZ235 and combination regimen with various mixed ratio. BEZ235 effectively inhibited leukemic cell growth with similar range of half maximal inhibitory concentration (IC50) values among different cell lines. Apoptosis was induced gradually as BEZ235 concentration increased, but significant level of apoptosis was not shown even at higher concentration beyond IC50 value. Then, AraC-resistant MV4-11 and THP-1 cell lines were chosen to investigate interaction between BEZ235 and AraC. Using CalcuSyn software based on Chou and Talalay analysis, Combination Index (CI) value was calculated in each combination regimen. Moderate to strong synergism was shown and it was well maintained as combination ratio of AraC versus BEZ235 gradually decreased from 20:1 to 1,000:1. BEZ235 reduced resistance to AraC when it was added as combination regimen, and significance of combination effect changed according to AraC concentration. When antileukemic effect was compared among combination regimens with different schedules, synergism was maximized when BEZ235 was pretreated before AraC administration. This means BEZ235 sensitizes leukemic cells to apoptotic effect of AraC.Genetic alteration in PI3K/Akt/mTOR pathway is an attractive target to investigate and dual pathway inhibitor BEZ235 has potential to maximize AML treatment through sensitization of leukemic cells to conventional drug. The results of this experiment suggest the possibility of designing early clinical trials for the therapy - AraC administration after dual inhibitor BEZ administration. An effective control of a signal transmission system using innovate new drugs along with an accurate understanding of a signal transmission system which is the target of therapy will establish a foothold for the development of AML therapy. Disclosures:No relevant conflicts of interest to declare.

The Phosphatidylinositol-3-Kinase Inhibitor BKM120 Impairs Proliferation and Induces Pro-Apoptotic Effects In Acute Myeloid Leukemia

Acute myeloid leukemia (AML) has been characterized by a growing number of recurrent genetic alterations, frequently causing the constitutive activation of signal transduction pathways, which result in enhanced blast proliferation and prolonged survival. Despite the increased understanding of AML biology, prognosis of these patients remains, generally, severe. Therefore, novel therapies targeted on aberrant signaling are now under evaluation. The phosphatidylinositol-3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway plays a pivotal role in the control of cell growth, proliferation and apoptosis of AML, and is often found constitutively activated. Currently, several inhibitors of this pathway, especially those inhibiting the activity of mTOR, have been investigated showing a limited efficacy due to the reactivation of upstream nodes. Since a novel PI3K inhibitor, BKM120, has been evaluated with encouraging results on solid tumors and lymphoid malignancies, we aimed in this study to investigate the pre-clinical activity of BKM120 on cell lines and primary AML samples. BKM120, kindly provided by Novartis, was tested on myeloid leukemia cell models - U937, MOLM13, OCIAML3 and KG1 - and on 4 primary AML samples at concentrations ranging from 0.5 to 5µM. Cell cycle changes and apoptosis induction were analyzed using the Acridine-Orange technique (AO) and the Annexin V (AnnV) binding assay. Intracellular signaling modulations induced by BKM120 were evaluated at different times (3h and 24h) by Western blot analysis. BKM120 exhibited dose- and time-dependent anti-proliferative and pro-apoptotic effects on the all analyzed leukemia cell models, although with different sensitivity (IC50s of 1.6, 2.4, 2.7 and 3.2μM for the U937, MOLM13, OCIAML3 and KG1 cell lines, respectively). On the most sensitive cell line U937, BKM120 in vitro exposure resulted, at 24h, in a significant cell growth reduction of 48.6% (p=0.006) and 73.9% (p=0.006), at 1 and 2µM, respectively. After 72h of treatment, AnnV positive cells significantly increased from 7.4% ± 0.03 (vehicle) to 43.5% ± 0.08 (p<0.001) and 89.4% ± 0.05 (p<0.001), at 1 and 2μM BKM120, respectively. On the less sensitive cell line KG1, the percentage of apoptotic cells reached 49.6% ± 0.1 and 56.6% ± 0.2 at 2 and 5μM BKM120, respectively. Since our previous data (Ricciardi MR et al., JMM, 2012) have been reported that the U937 cell line is characterized by PI3k/Akt/mTOR hyperactivation while the OCIAML3 cell line relies mostly on the Ras/Raf/MEK/Erk pathway, we evaluated the BKM120-induced intracellular signaling modulations on this two cell lines. After 3h of BKM120 exposure, the analysis of activation status of the PI3K/Akt/mTOR pathway revealed, on the U937, the downregulation of pAkt levels at 0.5µM associated with p70 and 4EBP1 dephosphorylation at 1µM. At variance, on the OCIAML3, both effects were achieved at higher concentrations (1 and 2µM, respectively), reflecting the different sensitivity to apoptosis of this model. A limited cytotoxicity (1.2 fold increase of apoptosis) was observed on normal PBMCs isolated from healthy volunteers following BKM120 exposure. In contrast, a pro-apoptotic activity was seen at 144h on the 4 primary AML cells (5.9, 3.9, 2.2 and 2.1 fold increase of apoptosis). Moreover, a mean increase of AnnV positive cells from 13.0% ± 0.06 (vehicle) to 23.2% ± 0.15, 25.6% ± 0.15, 32.9% ± 0.1 (p=0.02) and 40.3% ± 0.13 (p=0.02) was obtained on this cells following exposure to 0.5, 1, 2 and 5μM, respectively. In conclusion, our study demonstrates, at our knowledge for the first time, that the novel pan-class I PI3K inhibitor BKM120 impairs AML cells growth and proliferation inducing a pro-apoptotic activity and inhibiting the PI3K/Akt/mTOR signaling through dephosphorylation of the key components of this pathway. Disclosures:No relevant conflicts of interest to declare.

Characterization Of Leukemic Stem Cells In AML Cell Lines Using ALDH Staining

The clinical significance of LSCs has recently been confirmed by the finding that stem cell-like gene expression signatures can predict the clinical outcome of acute myeloid leukemia (AML) patients, what suggests that the targeted elimination of LSCs would be an efficient therapeutic approach in AML. LSCs are rather infrequent in patients' bone marrow, and hence are difficult to be identified and isolated. As an attempt to overcome such limitations, it has been previously shown that cell lines might be considered as attractive models aimed to better characterize LSC biological properties and their chemotherapy resistance mechanisms. Many attempts have been made to identify an immunophenotype that allow to discriminate between LSCs and normal hematopoietic stem cells (HSCs), but there is still controversy regarding the surface markers proposed so far. As a functional rather than immunological approach, the assessment of intracellular enzyme activities associated with the protection of primitive cells from oxidative insult during hematopoietic development has been proven as successful strategies, such as the use of Aldefluor reagent in the identification of AML LSCs. By taking advantage of the Aldefluor reagent in the field of stem cells research, we sought to identify LSC among the following AML cell lines, based on their high expression levels of the enzyme aldehyde dehydrogenase (ALDH): Kasumi-1, THP-1, MV-4;11, NB4 and OCI-AML3. Cells were stained with the Aldefluor reagent, following manufacturer's specifications, and cell subsets presenting high ALDH activity were identified in Kasumi-1 (13%), THP-1 (25%) and OCI-AML3 (12%). To assess if the Aldefluor staining was correlated to the expression of genes involved in stem cell maintenance or myeloid commitment, these cell lines were further sorted in two subpopulations, according to ALDH activity (ALDHhigh and ALDHlow/int), and the respective expression levels of the following genes were evaluated by RQ-PCR: CEBPA, BMI-1, NOTCH-1, C-MYC, HOXA9, E2F1, NANOG and OCT3/4. The delta-delta Ct method was used to determine the fold change in target genes between ALDHhigh and ALDHlow/int subpopulations. CEBPA, BMI-1 and NOTCH-1 genes were upregulated (9, 2.3 and 2 fold, respectively) in Kasumi-1 ALDHhigh cells when compared to ALDHlow/int, whereas C-MYC and E2F1 were downregulated (0.3 and 0.5 fold, respectively). Little is known about the physiological role of CEBPA in adult HSC biology, though it has been described as a central determinant in the switch from fetal to adult HSCs. Bmi-1 has been shown to be essential for the generation of self-renewing adult HSCs. Despite the roles of Notch signaling on myeloid development and AML are poorly understood, it can promote self-renewal, and induce growth arrest and apoptosis in hematopoietic stem cells. Some molecular mechanisms responsible for self-renewal, like Bmi-1 and Notch signaling pathways, were found to be shared by both HSC and LSC, suggesting that a higher expression of both genes in ALDHhigh cells would be in accordance with stemness properties. In line with these results, lower levels of C-MYC and E2F1 expression in ALDHhigh cells would suggest that they are somehow more quiescent than their ALDHlow/int counterparts. No significant differences were observed on the target genes expression levels among the other cell lines studied. In conclusion, our results reveal an important correlation between ALDH activity and the expression of genes associated with stemness properties in identifying stem cells in Kasumi-1 cell line, suggesting that like KG-1 cell line, which is also CD34-enriched, Kasumi-1 would be considered as a valuable model to better understand AML stem cells properties. Disclosures:No relevant conflicts of interest to declare.