Cultured Leukemia Cells Research Articles

Metabolism of Acute Myeloid Leukemia Cell Lines Alters with Passage in 2D Culture and Remains Stable in 3D

Acute Myeloid Leukemia (AML) is a heterogeneous and complex malignancy of the blood and bone marrow (BM). Current treatment options only cure 40% of patients under the age of 65 years and, over the age of 65 years only 10% will be cured. Hence, there is great urgency to further understand pathogenesis, optimize treatment and identify markers for diagnosis, prognosis and targets for novel therapies.It is increasingly evident that metabolism and BM microenvironment are key factors in AML pathogenesis and progression - entities that cannot be accurately investigated within current in vitro methods of two-dimensional (2D) culture. The metabolome is the final downstream product of gene transcription that also responds dynamically to its environment. Metabolomics is an established technique to identify biomarkers with prognostic relevance, such as 2-hydroxygluterate, an onco-metabolite derived from IDH mutations in AML. Targeting metabolic pathways such as oxidative phosphorylation, glycolysis and specific mutations that affect metabolism such as isocitrate dehydrogenase are currently being assessed in clinical trials. An in vitro system that could more accurately represent the in vivo BM may help optimise these metabolically-targeted regimens. Here we show that a previously established three-dimensional (3D) culture within our laboratory recapitulates elements of BM structure enabling long-term culture of AML cell lines without disrupting metabolism, as is the case with serial passage in 2D culture. Metabolism was assessed using gas chromatography-mass spectrometry (GC-MS) metabolomics analysis which reflects the phenotype of the leukemic cell lines.Porous polyurethane scaffolds were fabricated using dioxin by thermally induced phase separation as previously described; scaffolds have a pore size of 100-250µm and a porosity of 90-95% cut to 0.5cm3, coated with type I collagen and seeded with 0.5x106cells of either K562 (erythroleukemia) or HL-60 (promyelocytic leukemia) cells, after expansion in 2D culture. In parallel, 2D cultures of the same cell lines were maintained using passage every 2 days. K562 and HL-60 cells were cultured in IMDM, 1% penicillin and streptomycin and 20% or 10% foetal bovine serum, respectively. Serial sampling occurred at 4 time points over 21 days from 10 scaffolds of each condition. Cells were extracted from the 3D scaffolds using two techniques - needle extraction or TrypLE express (Thermo Fisher Scientific) to assess whether these physical or chemical extraction methods disrupt the metabolome. Once cells were extracted, they were suspended in methanol at 1x106cells/mL cold methanol for metabolite quenching; metabolites were subsequently extracted with methanol/water and derivatised with Methoxamine and N-Methyl-N-(trimethylsilyl)trifluoroacetamide. Metabolomics analysis was then performed using a Shimadzu QP2010 Ultra GC-MS machine detecting 138 metabolites including those of pertinent pathways: glycolysis, tricarboxylic acid, pentose phosphate, urea, glutaminolysis and amino acids. Bioinformatics analysis was performed with MeV TM4(http://mev.tm4.org).Using unsupervised clustering techniques, including hierarchical clustering and principal component analysis, we identified that the metabolome of TrypLE express and needle-extracted cells from 3D scaffolds have a similar metabolic signature and group closely with each other as well as with the metabolome of the seeded cells (day 0); they do not vary significantly over the 21 days of culture. Conversely, with each passage, the metabolomes of the 2D-cultured cells differ to those of day 0 and vary to each other over the same 21-day period.These results highlight limitations in the use of 2D cultures to address AML biology as metabolic changes with passage reflect change in phenotype. Based on these findings, we conclude that 3D cultures provide a more stable environment for leukemic cell culture and assessment of leukemia biology, irrespective of method of cell extraction. The 3D culture platform is more suited than standard 2D in vitro cultures to investigate metabolism, microenvironment and drug targets in AML. DisclosuresNo relevant conflicts of interest to declare.

Novel flavagline-like compounds with potent Fli-1 inhibitory activity suppress diverse types of leukemia.

E26 transformation-specific (ETS) gene family contains a common DNA-binding domain, the ETS domain, responsible for sequence-specific DNA recognition on target promoters. The Fli-1 oncogene, a member of ETS gene family, plays a critical role in hematopoiesis and is overexpressed in diverse hematological malignancies. This ETS transcription factor regulates genes controlling several hallmarks of cancer and thus represents an excellent target for cancer therapy. By screening compounds isolated from the medicinal plant Dysoxylum binectariferum in China, we identified two chemically related flavagline-like compounds including 4'-demethoxy-3',4'-methylenedioxyrocaglaol and rocaglaol that strongly inhibited Fli-1 transactivation ability. These compounds altered expression of Fli-1 target genes including GATA1, EKLF, SHIP1, and BCL2. Consequently, the flavagline-like compounds suppressed proliferation, induced apoptosis, and promoted erythroid differentiation of leukemic cells in culture. These compounds also suppressed erythroleukemogenesis in vivo in a Fli-1-driven mouse model. Mechanistically, the compounds blocked c-Raf-MEK-MAPK/ERK signaling, reduced phosphorylation of eukaryotic translation initiation factor 4E (eIF4E), and inhibited Fli-1 protein synthesis. Consistent with its high expression in myelomas, B-cell lymphoma, and B chronic lymphocytic leukemia (B-CLL), pharmacological inhibition of Fli-1 by the flavagline-like compounds or genetic knock-down via shRNA significantly hindered proliferation of corresponding cell lines and patients' samples. These results uncover a critical role of Fli-1 in growth and survival of various hematological malignancies and point to flavagline-like agents as lead compounds for the development of anti-Fli-1 drugs to treat leukemias/lymphomas overexpressing Fli-1.

A selective inhibitor of the Polo-box domain of Polo-like kinase 1 identified by virtual screening

Polo-like kinase 1 (PLK1), a member of the Polo-like kinase family, plays an important regulatory role in mitosis and cell cycle progression. PLK1 overexpression is correlated with tumourigenesis and poor prognosis in cancer patients. Therefore, the identification of novel compounds that inhibit PLK1 would provide attractive therapeutic approaches. Although some PLK1 kinase inhibitors have been developed, their application has been limited by off-target effects. PLK1 contains a regulatory domain named the Polo-box domain (PBD), which is characteristic only for the Polo-like kinase family. This domain represents an alternative therapeutic target with higher selectivity for PLK1. In this study, we applied in silico virtual drug screening, fluorescence polarization and microscale thermophoresis to identify new scaffolds targeting the PBD of PLK1. One compound, 3-{[(1R,9S)-3-(naphthalen-2-yl)-6-oxo-7,11-diazatricyclo[7.3.1.02,7]trideca-2,4-dien-11-yl]methyl}benzonitrile (designated compound (1)), out of a total of 30,793 natural product derivatives, inhibited the PLK1 PBD with high selectivity (IC50: 17.9 ± 0.5 µM). This compound inhibited the growth of cultured leukaemia cells (CCRF-CEM and CEM/ADR5000) and arrested the cell cycle in the G2/M phase, which is characteristic for PLK1 inhibitors. Immunofluorescence analyses showed that treatment with compound (1) disrupted spindle formation due to the aberrant localization of PLK1 during the mitotic process, leading to G2/M arrest and ultimately cell death. In conclusion, compound (1) is a selective PLK1 inhibitor that inhibits cancer cell growth. It represents a chemical scaffold for the future synthesis of new selective PLK1 inhibitors for cancer therapy.

A novel extracellular matrix-based leukemia model supports leukemia cells with stem cell-like characteristics

Acute myeloid leukemia (AML) relapse results from the survival of chemotherapy-resistant and quiescent leukemia stem cells (LSC). These LSCs reside in the bone marrow microenvironment, comprised of other cells and extracellular matrix (ECM), which facilitates LSC quiescence through expression of cell adhesion molecules. We used decellularized Wharton’s jelly matrix (DWJM), the gelatinous material in the umbilical cord, as a scaffolding material to culture leukemia cells, because it contains many components of the bone marrow extracellular matrix, including collagen, fibronectin, lumican, and hyaluronic acid (HA). Leukemia cells cultured in DWJM demonstrated decreased proliferation without undergoing significant differentiation. After culture in DWJM, these cells also exhibited changes in morphology, acquiring a spindle-shaped appearance, and an increase in the ALDH+ cell population. When treated with a high-dose of doxorubicin, leukemia cells in DWJM demonstrated less apoptosis compared with cells in suspension. Serial colony forming unit (CFU) assays indicated that leukemia cells cultured in DWJM showed increased colony-forming ability after both primary and secondary plating. Leukemia cell culture in DWJM was associated with increased N-cadherin expression by flow cytometry. Our data suggest that DWJM could serve as an ECM-based model to study AML stem cell-like cell behavior and chemotherapy sensitivity.

Design, synthesis and antineoplastic activity of novel hybrids of podophyllotoxin and indirubin against human leukaemia cancer cells as multifunctional anti-MDR agents

To overcome cancer drug resistance, in present study, a series of podophyllotoxin-indirubin hybrids were designed, synthesized, and evaluated for anticancer efficacy against two human chronic myeloid leukemia cell cultures. Among them, compound Da-1 was the most potent in resistent K562/VCR cells with an IC50 value of 0.076 ± 0.008 μM. Preliminary mechanism studies showed that Da-1 significantly induced apoptosis and cell cycle arrest at the G2 phase. Decrease in mitochondrial membrane potential, accompanied by activated PARP cleavage, was observed in K562/VCR cells after incubation with Da-1. Meanwhile, Da-1 caused the accumulation of intracellular ROS, regulated JNK and AKT signaling, and down-regulated the expression levels of P-gp and MRP1 proteins. Importantly, Western blotting revealed that Da-1 could induce K562/VCR cells autophagy, by increasing the levels of Beclin1 and LC3-II. Finally, Da-1 could disrupt microtubule organization, and binding mode to tubulin was investigated by using molecular modeling. Together, Da-1 was a novel hybrid with potent antiproliferative activity and might be a promising agent for the treatment of drug-resistant leukemia cancer.

Novel N-mustard-benzimidazoles/benzothiazoles Hybrids, Synthesis and Anticancer Evaluation.

Bendamustine, an N-mustard-benzoimidazole hybrid conjugate, was recently approved for the treatment of chronic lymphocytic leukemia. However, the short half-life of bendamustine may limit its clinical applications. The purpose of this study is to design and synthesize compounds with a more favorable pharmacokinetic profile. We synthesized a series of hybrid molecules comprising a phenyl N-mustard moiety and benzothiazole or benzimidazole scaffold linked via a urea linker and evaluated their antitumor activity and plasma stability. We revealed that these agents exhibited significant cytotoxicity against a panel of human lymphoblastic leukemia and human solid tumor cells in culture. Human lymphoblastic leukemia CCRM-CEM cells were the most sensitive to the tested compounds. In general, the new hybrids were as potent as cisplatin, but significantly more cytotoxic than bendamustine. Phenyl N-mustard-benzothiazole compound 27d and phenyl N-mustardbenzimidaloe compound 32b possessed significant cytotoxicity and led to apoptotic death in the treated tumor cells. These two agents were able to induce DNA interstrand cross-linking and arrested cell cycle progression at the G2/M phase. Furthermore, we showed that these new hybrids were more chemically stable than bendamustine in rat plasma. Our results suggest that conjugation of phenyl N-mustard pharmacophore at C6 of benzimidazole or at C8 of the benzothiazole ring via a urea linker is likely an approach to increase the chemical stability and bioavailability. Highlights ⇒ Series of benzimidazoles and benzothiazoles linked to N-mustard were synthesized. ⇒ The newly synthesized derivatives induced DNA interstrand cross-links. ⇒ These derivatives induced cell cycle arrest in the G2/M phase and triggered apoptosis in H460 cells. ⇒ The new compounds are more cytotoxic than bendamustine. ⇒ The new compounds were chemically more stable than bendamustine in rat plasma.

Structure-activity studies on N-Substituted tranylcypromine derivatives lead to selective inhibitors of lysine specific demethylase 1 (LSD1) and potent inducers of leukemic cell differentiation

FAD-dependent lysine-specific demethylase 1 (LSD1) is overexpressed or deregulated in many cancers such as AML and prostate cancer and hence is a promising anticancer target with first inhibitors in clinical trials. Clinical candidates are N-substituted derivatives of the dual LSD1-/monoamine oxidase-inhibitor tranylcypromine (2-PCPA) with a basic amine function in the N-substituent. These derivatives are selective over monoamine oxidases. So far, only very limited information on structure-activity studies about this important class of LSD1 inhibitors is published in peer reviewed journals. Here, we show that N-substituted 2-PCPA derivatives without a basic function or even a polar group are still potent inhibitors of LSD1 in vitro and effectively inhibit colony formation of leukemic cells in culture. Yet, these lipophilic inhibitors also block the structurally related monoamine oxidases (MAO-A and MAO-B), which may be of interest for the treatment of neurodegenerative disorders, but this property is undesired for applications in cancer treatment. The introduction of a polar, non-basic function led to optimized structures that retain potent LSD1 inhibitors but exhibit selectivity over MAOs and are highly potent in the suppression of colony formation of cultured leukemic cells. Cellular target engagement is shown via a Cellular Thermal Shift Assay (CETSA) for LSD1.

Abstract 2139: The effects of cyclodipeptide on the transport of doxorubicin and its cytotoxicity on tumor cell

Abstract Purpose: Chemotherapy using antitumor agents plays an important role in clinical cancer therapy. Among the treatments involving antitumor agents, the enhancement of antitumor activity was observed by combined chemotherapy. We have shown previously that some amino acids included theanine, taurine and anserine et al. as food components, increased doxorubicin (DOX) induced antitumor effect in vitro and in vivo. These effects by combined amino acids have depended on the increased DOX concentration in the tumor cells. Furthermore, these suppressions of DOX efflux by theanine and taurine were caused by the inhibitions of glutamate transporter and taurine transporter, respectively. The effect of anserine on DOX influx might connect with dipeptide transporter. Thus, other amino acid and unique peptides may act to DOX transport. In this study, the effects of cyclodipeptide on the transport of DOX and its cytotoxicity on tumor cell were clarified. Methods: Cyclo-leucine-proline (cyclo-Leu-Pro), cyclo-phenylalanine-proline (cyclo-Phe-Pro) and cyclo-glycine-proline (cyclo-Gly-Pro) as cyclodipeptide was used in this study. In transport of DOX, DOX influx or efflux was determined with combined cyclodipeptide in M5076 ovarian sarcoma cells or P388 leukemia cells. DOX concentration in tumor cell was measured using spectrofluorometer (Ex. 470 nm, Em. 580 nm). The cytotoxic study of DOX with combined cyclodipeptide was performed using WST-8. Results and Discussion: Cyclo-Phe-Pro and cyclo-Gly-Pro did not affect DOX influx and efflux in M5076 ovarian sarcoma cells and P388 leukemia cells. By combined cyclo-Leu-Pro, DOX influx into both M5076 ovarian sarcoma cells and P388 leukemia cells changed, compared to that of DOX only group. Into M5076 ovarian sarcoma, cyclo-Leu-Pro had strong effect in increased DOX influx in particular. And cyclo-Leu-Pro showed a tendency to suppress DOX efflux from M5076 ovarian sarcoma. In this studies of influx and efflux, concentration of cyclo-Leu-Pro existed optimum amount for increased DOX concentration in tumor cells. It is expected that cyclo-Leu-Pro induces the increment of DOX concentration in the tumor in vivo. DOX had the cytotoxic effects on M5076 ovarian sarcoma cells or P388 leukemia cells in culture. The combined cyclo-Leu-Pro with DOX increased cytotoxic effect, compared to that in the DOX alone group. In conclusion, cyclo-Leu-Pro has increased effect on DOX influx and inhibited effect on DOX efflux, and DOX induced cytotoxicity. It is expected that the combined cyclo-Leu-Pro will improve cancer chemotherapy by DOX. Moreover, cyclo-Leu-Pro may be help to prevent adverse effect since it will be able to decrease DOX dose. Citation Format: Ikumi Sugiyama, Yasuyuki Sadzuka. The effects of cyclodipeptide on the transport of doxorubicin and its cytotoxicity on tumor cell [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2139. doi:10.1158/1538-7445.AM2017-2139

Cytogenetic and Biochemical Investigations of Cultured Leukemia Cells Exposed to Gliding Arc Discharges

Cytogenetic and Biochemical Investigations of Cultured Leukemia Cells Exposed to Gliding Arc Discharges

A screen for Fli-1 transcriptional modulators identifies PKC agonists that induce erythroid to megakaryocytic differentiation and suppress leukemogenesis.

The ETS-related transcription factor Fli-1 affects many developmental programs including erythroid and megakaryocytic differentiation, and is frequently de-regulated in cancer. Fli-1 was initially isolated following retrovirus insertional mutagenesis screens for leukemic initiator genes, and accordingly, inhibition of this transcription factor can suppress leukemia through induction of erythroid differentiation. To search for modulators of Fli-1, we hereby performed repurposing drug screens with compounds isolated from Chinese medicinal plants. We identified agents that can transcriptionally activate or inhibit a Fli-1 reporter. Remarkably, agents that increased Fli-1 transcriptional activity conferred a strong anti-cancer activity upon Fli-1-expressing leukemic cells in culture. As opposed to drugs that suppress Fli1 activity and lead to erythroid differentiation, growth suppression by these new Fli-1 transactivating compounds involved erythroid to megakaryocytic conversion (EMC). The identified compounds are structurally related to diterpene family of small molecules, which are known agonists of protein kinase C (PKC). In accordance, these PKC agonists (PKCAs) induced PKC phosphorylation leading to activation of the mitogen-activated protein kinase (MAPK) pathway, increased cell attachment and EMC, whereas pharmacological inhibition of PKC or MAPK diminished the effect of our PKCAs. Moreover, in a mouse model of leukemia initiated by Fli-1 activation, the PKCA compounds exhibited strong anti-cancer activity, which was accompanied by increased presence of CD41/CD61 positive megakaryocytic cells in leukemic spleens. Thus, PKC agonists offer a novel approach to combat Fli-1-induced leukemia, and possibly other cancers,by inducing EMC in part through over-activation of the PKC-MAPK-Fli-1 pathway.

The Central Nervous System Microenvironment Influences the Leukemia Transcriptome and Enhances Leukemia Chemo-Resistance

Central nervous system (CNS) relapse is a significant cause of treatment failure among patients with acute lymphoblastic leukemia (ALL). Isolated CNS relapse occurs in ~3-8% of children with leukemia and accounts for 30-40% of initial relapses in some clinical trials. In addition, CNS-directed therapies are associated with seizures, secondary neoplasms, encephalopathy, and long-term endocrine, developmental, neurovascular, and cognitive deficits. While many studies have demonstrated that interactions between leukemia cells and components of the bone marrow microenvironment influence leukemia development, maintenance, and chemo-resistance, the role of the CNS microenvironment in leukemia is less well studied.To investigate the influence of the CNS niche in leukemia, we asked whether the CNS niche could impart unique and functionally important gene expression changes in leukemia cells. We transplanted NALM-6 human, pre-B leukemia cells into NSG mice without prior irradiation to avoid perturbing the bone marrow and CNS niches. After systemic leukemia development the mice were euthanized and leukemia cells were isolated from the bone marrow and CNS microenvironments. Gene expression profiling of ~700 cancer-associated genes (NanoString® Technology) identified 36 leukemia genes differentially expressed (30 up-regulated and 6 down-regulated; fold change ≥ 2 and FDR<0.05) in leukemia cells in the CNS microenvironment relative to the bone marrow. Furthermore, functional annotation revealed the up-regulated genes were involved in known leukemia and cancer pathways including MAPK, RAS, and apoptosis.We elected to further examine the gene PBX1 as it is a transcription factor with known roles in hematopoiesis, leukemia, and cancer biology. PBX1 contributes to Evi-1 mediated leukemia development in a murine model of leukemia and is a partner with TCF3 in the t(1;19) translocation that occurs in ~5% of pre-B ALL. Interestingly, this translocation is also associated with an increased risk for CNS relapse. We confirmed PBX1 mRNA up-regulation in leukemia cells either isolated from the CNS or in leukemia cells co-cultured with CNS-derived, murine choroid plexus cells by quantitative PCR. Supporting the generalizability of these results, additional human B-cell leukemia lines SEM and REH also exhibited PBX1 mRNA up-regulation in leukemia cells isolated from the murine CNS niche. Western blots of NALM-6 and SEM leukemia cells isolated from the mouse CNS as well as from leukemia cells co-cultured ex vivo with choroid plexus cells also showed PBX1 up-regulation at the protein level. Finally, culture of leukemia cells in either choroid plexus cell conditioned media or human cerebral spinal fluid (CSF) had minimal effects on PBX1 protein levels, suggesting that direct cell contact, rather than a soluble factor(s), contributes to PBX1 up-regulation in leukemia cells.Following confirmation of PBX1 up-regulation in leukemia cells within the CNS microenvironment, we next ectopically expressed PBX1, or GFP as a control, in leukemia cells to identify functional consequences of PBX1 expression in leukemia cells. Leukemia cells expressing PBX1 exhibited decreased sensitivity to cytarabine relative to control cells as measured by both proliferation and apoptosis assays. Furthermore, shRNA targeting PBX1, but not control shRNA, prevented PBX1 up-regulation in leukemia cells when co-cultured with choroid plexus cells and modestly, but significantly, attenuated the ability of choroid plexus cells to protect leukemia cells from cyatabine-induced apoptosis. Finally, although PBX1 had no effect on leukemia proliferation, it caused enhanced colony-forming ability in semi-solid media, consistent with increased leukemia self-renewal properties. Together these results suggest PBX1 up-regulation in leukemia cells may contribute to both leukemia self-renewal properties and chemo-resistance in the CNS niche.In summary, we believe this work illustrates the unique and functionally important effect that the CNS microenvironment has on leukemia cells. More comprehensive analyses of the leukemia transcriptome, genome, and proteome in the CNS niche will build upon this foundation and provide a more detailed understanding of the role of the CNS niche in leukemia biology. Finally, this approach may identify targetable CNS niche-mediated mechanisms of leukemia chemo-resistance and self-renewal. DisclosuresNo relevant conflicts of interest to declare.

Bone Marrow Stromal Cell Mediated Resistance to Mertk Inhibition in Acute Leukemia

MERTK is a receptor tyrosine kinase of the TAM family (TYRO-3, AXL, MERTK) that is ectopically expressed in 30-50% of newly diagnosed pediatric acute lymphoblastic leukemia (ALL) patient samples and aberrantly expressed in 80-100% of pediatric and adult primary acute myeloid leukemia (AML) samples. MERTK inhibition mediated by shRNA or a small molecule inhibitor, MRX-2843, decreased colony-forming potential and induced apoptosis in leukemia cell cultures. Moreover, MERTK inhibition prolonged survival in mouse xenograft models of acute leukemia, but was not curative. In these models, treatment with MRX-2843 effectively reduced peripheral disease burden but was less effective in the bone marrow, suggesting a role for the bone marrow microenvironment in therapeutic resistance. Additionally, Gas6, a MERTK ligand, is a poor prognostic factor in AML, mediates increased resistance to cytotoxic chemotherapy in leukemia cells, and is expressed in the bone marrow.To determine the role of Gas6 produced by bone marrow stromal cells in mediating resistance to MERTK inhibition by MRX-2843, acute leukemia cell lines were cultured in the presence of a Gas6-producing fibroblast-like cell line (HS27) or bone marrow derived stromal cells (BMDSCs) from wild type or Gas6 knockout mice and induction of apoptosis and cell death was determined by flow cytometry after treatment with MRX-2843 or vehicle. Co-culture with the HS27 cell line significantly reduced cell death in Kasumi-1 AML cell cultures in response to treatment with 300nM MRX-2843 compared to leukemia cells alone (29.4% versus 60.5%, p=0.002). Similar results were observed in Nomo1 AML and 697 pre-B ALL cell cultures. To evaluate whether soluble factors mediated this protective effect, Kasumi-1 cells were cultured in HS27-conditioned medium in the presence or absence of MRX-2843. Interestingly, conditioned medium was not sufficient to provide protection from MRX-2843 induced apoptosis (86.0% vs. 85.5% in unconditioned medium). To more directly assess the role of Gas6, BMDSCs isolated from wild-type and Gas6 knockout C57Bl/6 mice were co-cultured with 697 leukemia cells and sensitivity to MRX-2843 was determined. BMDSCs from wild-type mice protected 697 leukemia cells from MRX-2843 induced cell death much more effectively than BMDSCs from Gas6 knockout mice (4.3% apoptotic and dead cells versus 72.4%, respectively).To investigate biochemical mechanisms of Gas6-mediated protection, Kasumi-1 AML cells were cultured with 200nM MRX-2843 or vehicle in the presence or absence of HS27 cells and expression and activation of MERTK, AXL, TYRO-3, and downstream signaling effectors STAT5, AKT, and ERK1/2 were determined by immunoblot. AXL was not expressed in Kasumi-1 cells with or without co-culture. Treatment with MRX-2843 mediated robust inhibition of MERTK activation indicated by reduced levels of phosphorylated protein in both the presence and absence of stromal cell co-culture. In contrast, activation of TYRO-3 was increased after treatment with MRX-2843 in leukemia cells co-cultured with HS27 stromal cells. Similarly, in the absence of co-culture MRX-2843 inhibited activation of STAT5, AKT, and ERK1/2. However, in the presence of HS27 cells there was robust activation of STAT5 that was sustained even after treatment with MRX-2843. In contrast, MRX-2843 inhibited activation of AKT and ERK1/2 in HS27 co-cultures, although higher doses were required.Together these data support a model whereby Gas6 produced by stromal cells mediates leukemia cell resistance to MERTK inhibition in the bone marrow by inducing activation of TYRO-3, thereby promoting downstream signaling and cell survival despite MERTK inhibition. Thus, combined treatment with MRX-2843 and a TAM ligand sink (eg MERTK-Fc), a TYRO-3 inhibitor, or a bone marrow mobilizing agent may be particularly effective therapeutic strategies. DisclosuresWang:Meryx, Inc: Equity Ownership, Patents & Royalties: MRX-2843. Frye:Meryx, Inc: Equity Ownership, Patents & Royalties: MRX-2843. Earp:Meryx, Inc: Equity Ownership, Patents & Royalties: targeting MERTK. DeRyckere:Meryx, Inc: Equity Ownership, Patents & Royalties: targeting MERTK. Graham:Meryx, Inc: Equity Ownership, Patents & Royalties: targeting MERTK.

Evaluation of Genetic Mutations in Plasma Exosomes As a Biomarker Tool for Detecting the Clonal Evolution of Leukemic Cells in Pediatric AML

Abstract Introduction: AML is the second most of pediatric leukemia with relapse in &gt;30% of the patients.Clonal evolution of rare primary leukemic cells that survived the initial therapy or gained additional mutations independent of therapy stress, could be the potential cause of relapse in pediatric AML.Therefore, sensitive and reliable methods to measure accurately the mutational shifts between diagnoses, during therapy and relapse could provide useful information on the disease progression.Exosomes are extracellular vesicles of 30-150nm in diameter that are released by both healthy and malignant cells. Exosomes derived from tumor cells or leukemia blasts have emerged as potential valuable biomarkers as they have been illustrated to feature disease specific protein, lipid and nucleic acid signatures that represent the pathological state of the respective cells. While leukemia cells release relatively higher amounts of exosomes compared to healthy cells, mutational profiling of exosomes could be more sensitive than analyzing rare leukemia sub-clone in thehematopoietic compartment. Methods: Secreted exosomes from conditioned media of K-562leukemia celllines and plasma of pediatric AML patients were isolated using differential ultracentrifugation at 100000g. Absolute DNA amount was quantified usingQuantiFluordsDNA System fromPromega. DNA deep sequencing was performed to analyze theexosomal DNA from K-562 cell line. The mutational profiling of the DNA from primary material, plasma and exosomes was performed using next generation sequencing platform, Illumina. Results: To establish the diagnostic and prognostic potential of exosomes in measuring leukemia associated mutations we isolated exosomes from the conditioned media of the cultured leukemia cell lineK-562. We used this cell line as model because it harbors the classical BCR-ABL translocation that is relevant in deciding treatment options in chronic myeloid leukemia. Analysis of DNA isolated from these exosomes revealed the presence of genomic double-stranded DNA (dsDNA) fragments. Using deep sequencing approach we could further detect the classical BCR-ABL translocation in exosomes. The presence of leukemia specific mutation in exosomes that is potentially derived from parental cells suggests the utility for exosomes in leukemia diagnosis and to forecast treatment response and relapse. In the next step, we analyzedexosomal dsDNA from plasma exosomes of primary pediatric AML patients. We compared the sensitivity of DNA associated from exosomes isolated from blood plasma of pediatric AML patients and compared it to free floating cell free DNA (cfDNA) in the plasma supernatant. In our study we used the plasma volume ranging from 350 microliters to 1ml from peripheral blood of AML patients at diagnosis and relapse (n=9). We performed DNA isolation from the starting plasma material and then compared it with the exosomes isolated. As control, DNA from the exosomes depleted fraction was also analyzed. Comparison of these three fractions (input plasma, pelleted exosomes and exosome depleted plasma) revealed a significant enrichment of dsDNA in the exosome fraction after ultracentrifugation in 7 out of 9 patients (Figure A). This suggests that isolation of exosomes from supernatant can enrich the amount of DNA and thereby the sensitivity of downstream mutational analysis in diagnostic. By next generation sequencing (NGS) (myeloid panel, Illumina) using the DNA materials from plasma supernatants and the respective exosomes we screened for AML associated mutations (n=7). We were able to detect the known primary mutation detected at diagnosis or relapse in both plasma and exosomes of 6 out of 7 patient material. In one patient the detection of mutations were not possible in both the plasma andexosomal fraction. Interestingly we could detect additional mutations like FLT-ITD (patient 2) or CUX1 (patient 4) only in the exosome fraction (Table B). Conclusion: These results suggest that the combined evaluation of DNA from plasma and exosome fraction could provide novel and additional information about the clonal hierarchy or evolution of AML, and if used for monitoring, might be of prognostic relevance. Disclosures Reinhardt: Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees; Jazz Pharma: Other: Travel Accomodation; Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees.

STIP Regulates ERK1/2 Signaling Pathway Involved in Interaction with PP1γ in Lymphoblastic Leukemia.

Sip1/Tuftelin Interacting Protein (STIP) is highly conserved from Caenorhabditis elegans to Homo sapiens and has essential biological functions. However, its function in leukemia remains unknown. Clinic samples and cell model were used in this article to investigate the expression of STIP in lymphoblastic leukemia. The functional research of STIP was performed in ARH-77 by siRNA transfection, immunofluorescence, cell count, cell cycle analysis, qRT-PCR, sub-cellular fractionation assays, immunoprecipitation and western blotting. Here, we found that STIP is more highly expressed in both clinical lymphoblastic leukemia samples and cultured leukemia cells than in normal samples. Knockdown of STIP in B lymphoblastic leukemia ARH-77 cells leads to S phase arrest, lower cell proliferation rates, and suppressed AKT and ERK1/2 signaling pathways. Interestingly, when protein phosphatase was inhibited by Calyculin A, STIP knockdown did not result in the dephosphorylation of p-ERK1/2, suggesting the dependence of STIP on protein phosphatase in the regulation of ERK1/2. Among those protein phosphatase inhibited by Calyculin A, PP1γ was found to interact with STIP proven by immunofluorescence and immunoprecipitation assays. The binding of STIP with PP1γ may decrease the phosphatase activity of PP1γ, resulting in hyper-activated ERK1/2 signaling. In summary, the high expression and activation effect on the ERK1/2 signaling of STIP in lymphoblastic leukemia suggest that STIP would be a potential therapy target or diagnosis marker for leukemia.

Establishment and Characterization of Long-Term Cultures Derived from Primary Acute Myeloid Leukemia Cells for HDAC Inhibitor Research.

Histone deacetylase (HDAC) inhibitors are promising drugs. These agents lead to growth inhibition, cell cycle arrest, premature senescence, and apoptosis of malignant cells. Aim of our studies was to determine the efficacy of HDAC inhibitors on the clinically most relevant population of human leukemic progenitor cells in vitro. We here present stroma-free long-term cultures (LTC) of primary acute myeloid leukemia (AML) cells as a useful system for drug sensitivity testing in functional assays. AML-LTC are established by isolating mononuclear cells from peripheral blood samples of AML patients followed by selection of CD34+ progenitor cells. AML-LTC cells can be maintained in liquid culture supplemented with cytokines and utilized for in vitro analyses to assess proliferation, apoptosis, expression of surface proteins or intracellular proteins and signal transduction, respectively.

Allee effect in leukemia

Allee effect is a phenomenon in ecology characterized by a correlation between population size or density and the mean individual fitness of population or species. It is consistent with density dependent phenomenon in biomedical field. Recently, Allee effect has been studied by theoretical biologist in detail for oncology biological research. Leukemia cells, which reside in organic microenvironment, show an obvious Allee effect. The authors compared Allee effect with density dependent growth of leukemia cells based on their work experience and literature data. No principle distinction was found between these two terms. In this paper, Allee effect in leukemia cell culture and leukemia, especially in minimal residual disease, will be discussed in the view of ecology. The association between Allee effect and leukemogenesis and relapse dynamics will also be explored in the future research. Key words: Leukemia; Minimal residual disease; Allee effect; Evolutionary medicine

Effect of cigarette smoke extract on phagocytosis of Staphylococcus aureus by macrophages

Staphylococcus aureus is one of the most important pathogen that causes community acquired and nosocomial infections worldwide. Phagocytosis by macrophages plays an important role in the first line defense against infections caused by S.aureus. On the other hand, the conducted studies have indicated that cigarette smoke has negative effects on both innate and acquired immune responses. The aim of this study was to investigate the effects of cigarette smoke on macrophage viability and their capacity of S.aureus phagocytosis. For this purpose THP-1 cell lines (human leukemic monocyte cell culture) were used and after the differentiation of the cells with PMA (phorbol myristate acetate) treatment, the macrophages were exposed to cigarette smoke extract (CSE) for 2- and 4-hours at concentrations of 1%, 5%, 10%, 25%, and 50%. Afterwards, the cells were stained with propidium iodide and the viability of the cells was analyzed by a flow cytometer. Two different methods were used to investigate the effect of CSE on the phagocytosis of S.aureus. The first one was the classical bacteriological method, in which macrophages were exposed to CSE for 2 hours in five different concentrations and were infected with 100 MOI (multiplicity of infection) S.aureus. After 1 hour of incubation, macrophages were lysed with PBS-0.1% Triton X-100 and plated on Luria-Bertani (LB) agar following serial dilutions. Newly formed colonies were counted and the number of bacteria phagocytosed were evaluated as colony forming units (CFU). The second method for the detection of phagocytosis was flow cytometric analysis in which SYBR(®) Green-labeled bacteria were used. To confirm that the macrophages were infected, bacteria were stained with SYBR(®) Green and macrophages were analyzed following infection via flow cytometry. Macrophages were exposed to 10% and 50% CSE and infected with bacteria stained with SYBR(®) Green. The level of phagocytosis was analyzed by flow cytometry in terms of median fluorescence intensity. Macrophage death rate was 24% and 30% following 2-hour exposure to 25% and 50% CSE, respectively, while 4-hour exposure increased death rate to 38% and 51%, respectively (p< 0.001). At 10% and higher concentrations of CSE, cell death increased with an average of 1.5-fold between 2- and 4-hour exposures (p< 0.05). Macrophages were successfully infected (99.8%) with SYBR(®) Green-stained S.aureus. Phagocytosis of S.aureus by macrophages decreased significantly upon exposure to 10% or more CSE concentrations (p< 0.0001). Median fluorescence intensity, which indicates phagocytosed bacterial cell number, decreased with no statistical significance when macrophages exposed to 10% and 50% CSE were infected with SYBR(®) Green-stained S.aureus. The results of this study revealed that, macrophage viability and phagocytosis of S.aureus were reduced depending on CSE concentration and time of exposure. In addition, it was shown that, SYBR(®) Green dye is a proper stain for the flow cytometric analysis of bacteria that were phagocytosed by macrophages.

18F]CFA as a clinically translatable probe for PET imaging of deoxycytidine kinase activity

Deoxycytidine kinase (dCK), a rate-limiting enzyme in the cytosolic deoxyribonucleoside (dN) salvage pathway, is an important therapeutic and positron emission tomography (PET) imaging target in cancer. PET probes for dCK have been developed and are effective in mice but have suboptimal specificity and sensitivity in humans. To identify a more suitable probe for clinical dCK PET imaging, we compared the selectivity of two candidate compounds-[(18)F]Clofarabine; 2-chloro-2'-deoxy-2'-[(18)F]fluoro-9-β-d-arabinofuranosyl-adenine ([(18)F]CFA) and 2'-deoxy-2'-[(18)F]fluoro-9-β-d-arabinofuranosyl-guanine ([(18)F]F-AraG)-for dCK and deoxyguanosine kinase (dGK), a dCK-related mitochondrial enzyme. We demonstrate that, in the tracer concentration range used for PET imaging, [(18)F]CFA is primarily a substrate for dCK, with minimal cross-reactivity. In contrast, [(18)F]F-AraG is a better substrate for dGK than for dCK. [(18)F]CFA accumulation in leukemia cells correlated with dCK expression and was abrogated by treatment with a dCK inhibitor. Although [(18)F]CFA uptake was reduced by deoxycytidine (dC) competition, this inhibition required high dC concentrations present in murine, but not human, plasma. Expression of cytidine deaminase, a dC-catabolizing enzyme, in leukemia cells both in cell culture and in mice reduced the competition between dC and [(18)F]CFA, leading to increased dCK-dependent probe accumulation. First-in-human, to our knowledge, [(18)F]CFA PET/CT studies showed probe accumulation in tissues with high dCK expression: e.g., hematopoietic bone marrow and secondary lymphoid organs. The selectivity of [(18)F]CFA for dCK and its favorable biodistribution in humans justify further studies to validate [(18)F]CFA PET as a new cancer biomarker for treatment stratification and monitoring.

Predictive Drug Sensitivity Testing with Patient-Derived Cultured Leukemia Cells

Predictive Drug Sensitivity Testing with Patient-Derived Cultured Leukemia Cells

Adrenomedullin in the growth modulation and differentiation of acute myeloid leukemia cells

Adrenomedullin (ADM) is a regulatory peptide endowed with multiple biological effects, including the regulation of blood pressure, cell growth and innate host defence. In the present study, we demonstrated that ADM signaling could be involved in the impaired cellular differentiation of myeloid leukemia cells to mature granulocytes or monocytes by modulating RAMPs/CRLR expression, PI3K/Akt cascade and the ERK/MAPK signaling pathway. When exogenously administered to in vitro cultures of HL60 promyelocytic leukemia cells, ADM was shown to exert a strong proliferative effect with minimal upregulation in the expression level of monocyte antigen CD14. Notably, the experimental inhibition of ADM signaling with inhibitor ADM22-52 promoted a differentiative stimulation towards monocytic and granulocytic lineages. Moreover, based on the expression of CD31 relative to CD38, we hypothesized that an excess of ADM in bone marrow (BM) niche could increase the transendothelial migration of leukemia cells while any inhibitory event of ADM activity could raise cell retention in hyaluronate matrix by upregulating CD38. Taken into consideration the above evidence, we concluded that ADM and ADM22-52 could differently affect the growth of leukemia cells by autocrine/paracrine mechanisms and may have clinical relevance as biological targets for the intervention of tumor progression.