Kasumi-1 Cells Research Articles

Low-Dose Triptolide Enhanced Activity of Idarubicin Against Acute Myeloid Leukemia Stem-like Cells Via Inhibiting DNA Damage Repair Response.

Leukemia stem cells (LSCs) are considered to be the root of relapse for acute myeloid leukemia (AML). Conventional chemotherapeutic drugs fail to eliminate LSCs. Therefore, new therapeutic strategies eliminating LSCs are urgently needed. Our results showed that low-dose Triptolide (TPL) enhanced the anti-AML activity of Idarubicin (IDA) in vitro against LSC-like cells (CD34 + CD38- KG1αand CD34 + CD38- kasumi-1 cells) and CD34+ primary AML cells, while sparing normal cells. Inspiringly, the combination treatment with low-dose TPL and IDA was also effective against CD34 + blasts from AML patients with FLT3-ITD mutation, which is an unfavorable risk factor for AML patients. Moreover, the combination of TPL and IDA induced a remarkable suppression of human leukemia growth in a xenograft mouse model. Mechanistically, the enhanced effect of low dose TPL on IDA against LSCs was attributed to inhibiting DNA damage repair response. Thus, our study may provide a theoretical basis to facilitate the development of a novel LSCs-targeting strategy for AML.Graphical abstract.

Boswellia dalzielii-Mediated Silver Nanoparticles Inhibited Acute Myeloid Leukemia (AML) Kasumi-1 Cells by Inducing Cell Cycle Arrest.

Background Acute myeloid leukemia (AML) persists to be a major health problem especially among children as effective chemotherapy to combat the disease is yet to be available. Boswellia dalzielii is a well-known herb that is traditionally used for treatment and management of many diseases including degenerative diseases. In this study, silver nanoparticles were synthesized from the phytochemicals of B. dalzielii stem bark aqueous extract. The silver nanoparticles were characterized by carrying out Fourier Transform Infrared (FTIR) spectroscopy, Energy Filtered Scanning Electron Microscopy (FESEM), X-ray diffraction, and Dynamic Light Scattering (DLS) analyses. Antioxidant capacity of the nanoparticles was evaluated using 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, and the antiproliferative effect of the nanoparticles on Kasumi-1 leukemia cells was investigated using PrestoBlue assay. Flow cytometry analysis was performed to observe the effect of the nanoparticles on the leukemia cell cycle progression. Results Our findings revealed that the synthesized silver nanoparticles were formed from electrons of the plant phytochemicals which include aromatic compounds, ethers, and alkynes. FESEM analysis revealed that the sizes of the nanoparticles range from 12 nm to 101 nm; however, DLS analysis estimated a larger average size of the nanoparticles (108.3 nm) because it measured the hydrodynamic radii of the nanoparticles. The zeta potential of the nanoparticles was −16 nm, and the XRD pattern of the nanoparticles has distinct peaks at 38.02°, 42.94°, 64.45°, 77.20°, and 81.47°, which is typical of face-centered cubic (fcc) structure of silver. The Trolox Equivalence Antioxidant Capacity (TEAC) of the nanoparticles was estimated to be 300.91 μM Trolox/mg silver nanoparticles. The nanoparticles inhibited Kasumi-1 cell proliferation. The half minimal inhibitory concentrations (IC50s) that inhibited Kasumi-1 cell proliferation are 49.5 μg/ml and 13.25 μg/ml at 48 and 72 hours, respectively. The nanoparticles induced cell cycle arrest in the Kasumi-1 cells at S (5% increase) and G2/M (3% increase) phases. Conclusion The nanoparticles synthesized from the stem bark extract of B. dalzielii inhibit the growth of Kasumi-1 leukemia cells by activating cell cycle arrest; thus, they are potential antileukemic agents.

Abstract 2887: Bispecific antibody drug conjugates targeting CD7 and CD33 for the treatment of acute myeloid leukemia

Abstract Phenotyping of AML patient samples shows that CD33, a myeloid cell marker, and CD7, a lymphoid cell marker, are specifically co-expressed in 25% of AML cases. Development of a bispecific antibody drug conjugate (ADC) dependent on co-expression of CD7 and CD33 may lead to an ADC with better tumor-cell selectivity than the anti-CD33 ADC, gemtuzumab ozogamicin. Anti-CD7 and -CD33 Fabs were expressed in HEK293 cells, and affinities assessed using surface plasmon resonance. Chemically cross-linked anti-CD7, anti-CD33 Bi-Fabs were produced using click chemistry via an individual cysteine residue in each CH1 domain. Bi-Fabs were conjugated to valine-citrulline-monomethylauristatin E (vcMMAE) and maleimidocaproyl monomethylauristatin F (mcMMAF) at native cysteine residues. Bi-Fab drug conjugates were assessed for cytotoxicity against HNT-34 and Kasumi-3 cells (CD7+CD33+), and assessed for selectivity against primary human T cells and myeloid progenitor cells, using monospecific Bi-Fab and gemtuzumab drug conjugates with equivalent drug-antibody ratios as controls. The activity of the lead Bi-Fab, BVX130-mcMMAF, was assessed in a subcutaneous (s.c.) xenograft model in SCID mice implanted with HNT-34 cells. Animals (n=10/group) received BVX-130-mcMMAF (10mg/kg twice weekly i.v. for 4 weeks), cytarabine at its maximum tolerated dose (20mg/kg, 5 days on 2 days off i.p. for 3 weeks), or vehicle control for 4 weeks. The wild-type Fabs had Kd values of 3.6nM (anti-CD7 Fab) and 1.6 nM (anti-CD33 Fab) for their respective antigens. The resulting anti-CD7, anti-CD33 Bi-Fab MMAE conjugate (BVX100-vcMMAE) was highly cytotoxic to HNT-34 and Kasumi-3 cells with IC50 values of 0.20±0.04nM and 0.20±0.03nM respectively, compared to IC50 values of 1.3nM and 1.2nM for gemtuzumab MMAE. BVX100-vcMMAE had 5-fold lower cytotoxicity towards Jurkat cells (CD7+CD33−) and >10-fold margins relative to MV4.11 and SHI-1 (CD7−CD33+) cells whereas corresponding homo-dimeric Bi-Fabs were non-selective. Affinity reduction of the CD7 Fab resulted in Bi-Fab drug conjugates with similar activity and improved selectivity. One such Bi-Fab, BVX130mcMMAF, had no measurable cytotoxicity to resting T-cells, and did not inhibit colony formation in a GM-CFU assay, at concentrations up to 10nM. Treatment of mice carrying s.c. HNT-34 tumors with BVX130-mcMMAF resulted in Tumor Growth Inhibition (TGI) values of 100% (98% regression, p<0.001) and 100% (97% regression, p<0.001) at days 21 and 28 respectively, with three animals tumor-free at the end of dosing. Treatment with cytarabine resulted in TGI of 100% (47% regression, p<0.001) at day 21. No adverse effects were observed in the BVX130-mcMMAF treatment group. Anti-CD7, anti-CD33 Bi-Fab drug conjugates are highly cytotoxic to CD7+CD33+ cells in vitro and in vivo, and have substantially greater selectivity than monospecific drug conjugates for such cells, supporting the development of bispecific antibody drug conjugates targeting CD7 and CD33 for the treatment of CD7+/CD33+ AML. Citation Format: Richard Bethell, Cath Eberlein, Victoria Pollard, Theonie Georgiou, George Orphanides, Lorraine Mooney, Jane Kendrew, Tiffany Daniels. Bispecific antibody drug conjugates targeting CD7 and CD33 for the treatment of acute myeloid leukemia [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2887.

HDAC Inhibitor LBH589 Suppresses the Proliferation but Enhances the Antileukemic Effect of Human γδT Cells

γδT cells have potent effects on hematological malignancies, and their functions can be regulated by anti-tumor agents. Histone deacetylase inhibitors (HDACis) not only have antileukemic activity on leukemia but also affect immune cells during therapeutic application. In this in vitro study, we showed that LBH589, a pan-HDACi, impaired the proliferation of human γδT cells, as well as their proportions in peripheral blood mononuclear cells (PBMCs). At the specific concentration, LBH589 induced significant antileukemic activity of γδT cells against the HL-60 cells and Kasumi cells in a dose-dependent manner. However, the expression levels of activating receptor and molecules, as well as interferon-γ (IFN-γ) expression on γδT cells, were not affected by LBH589. After treatment with LBH589 for indicated times, extracellular-regulated protein kinase (ERK), Akt, and c-Jun N-terminal kinase (JNK) signaling pathways in γδT cells were not activated. In contrast, a stronger expression of Notch was observed and sustained for 72 h. Inhibition of Notch signaling by FLI-06, the γ-secretase inhibitor, significantly reversed the enhanced antileukemic ability of γδT cells induced by LBH589. For the first time, our investigations demonstrate that LBH589 can inhibit proliferation of γδT cells but facilitate their antileukemic effects via activation of Notch signaling.

The Cytotoxic Effects of Moringa oleifera Leaf Extract and Silver Nanoparticles on Human Kasumi-1 Cells.

BackgroundMoringa oleifera, commonly known as “moringa”, is widely cultivated in tropical and subtropical regions across the globe. Extensive studies have shown that various parts of the moringa tree exhibit anti-cancer properties. This study determined the effects of sequential moringa leaf extracts and silver nanoparticles synthesized from moringa leaf extract on Kasumi-1 leukemia cells.Methods and ResultsDried moringa leaf powder was sequentially extracted with the assistance of ultrasound starting with absolute ethanol, followed by 50% ethanol, and finally, deionized water. The aqueous extract was utilized to synthesize silver nanoparticles. The optimum conditions to generate moringa silver nanoparticles (MO-AgNPs) were eight hours of incubation at 60°C with 1 mM silver nitrate and 1% moringa aqueous extract from sequential extraction. The three extracts and MO-AgNPs were used to treat Kasumi-1 cells for 24, 48, 72 hours with concentrations ranging from 400 to 12.5 µg/mL, while cell viability was determined with 3(4, 5-dimethythiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. After 72 hours of treatment, the moringa leaf absolute ethanol extract displayed the strongest inhibitory effects on Kasumi-1 cells with IC50 of 10 µg/mL, in comparison to moringa leaf 50% ethanol extract (25 µg/mL) and aqueous extract (>400 µg/mL). Interestingly, MO-AgNPs exhibited the strongest cytotoxic effects on Kasumi-1 cells with an IC50 of 7.5 µg/mL. Cytotoxic study on normal CD34+ cells treated with up to 50ug/mL of either MO-AgNPs or ethanol extract still had more than 80% cell viability indicating that the treatments have selective cytotoxicity against the cancer cells. Morphological studies of Kasumi-1 cells treated with IC50 of moringa leaf ethanolic extract and MO-AgNPs show a lot of shrinking, dying cells and cell debris. Cell cycle studies displayed an increase in cells at the G1 phase for ethanol leaf extract, while MO-AgNPs caused cell cycle arrest at the S phase after treatment with IC50 dose for 24 hours. Moringa leaf ethanol extract and the nanoparticles induced apoptosis in Kasumi-1 cells as shown by annexin V – FITC assays. Gene expression analysis by qPCR verified these outcomes, as the moringa leaf ethanol extract led to significant upregulation of proapoptotic gene caspase 8, whereas the MO-AgNPs caused a significant increase of proapoptotic protein BID.ConclusionThis study reveals that moringa ethanolic leaf extract and MO-AgNPs induced potent antiproliferative effects in Kasumi-1 cells by apoptosis.

3099 – REPRESSION OF TBL1XR1 BY MIR-130A REINFORCES THE ABERRANT MOLECULAR PROGRAM IN T(8,21) AML

Deregulation of self-renewal and differentiation programs are central to the pathogenesis of hematologic malignancies. MicroRNAs (miRNAs) represent a large class of post-transcriptional regulators that mediate repression of multiple target mRNAs and are frequently deregulated in acute myeloid leukemia (AML). To identify miRNA(s) that play a functional role in human hematopoiesis, we performed an in vivo competitive repopulation screen in which candidate miRNAs were over-expressed (OE) in human CD34+CD38- umbilical cord blood (CB) cells and subsequently transplanted into immune-deficient mice for 24 weeks. miR-130a was shown to enhance long-term hematopoietic reconstitution and chosen for further investigation. Enforced expression of miR-130a conferred a competitive repopulation advantage and induced an expansion of HSPC in xenograft assays. Secondary transplantation at limiting dilution revealed a 10-fold increase in HSC frequency, consistent with a role of miR-130a in enhancing HSC self-renewal. Label-free semi-quantitative proteomics in human HSPC combined with GSEA identified chromatin remodeling as the most significant pathway repressed by miR-130a. TBL1XR1, a component of the nuclear receptor corepressor (NCoR) complex that mediates the exchange of corepressors for coactivators, and CBFb, the β subunit of RUNX1, were among the most repressed miR-130a targets. Interestingly, interrogation of AML datasets revealed elevated expression of miR-130a in t(8,21) AML characterized by the fusion of AML1(RUNX1) and ETO genes. High miR-130a expression was correlated with poor patient overall survival. AML1-ETO recruits the aforementioned NCoR complex to repress RUNX1 target genes, thus preventing myeloid differentiation. To test whether miR-130a controls the AML1-ETO molecular program via repression of TBL1XR1, we tested the effect of miR-130a knock-down in Kasumi cell line and patient samples with the t(8,21). Notably, miR-130a KD resulted in differentiation of transduced cells and increased protein levels of TBL1XR1. Immunoprecipitation of AML1-ETO following miR-130a KD revealed loss of NCoR and CBFβ. Thus, we have uncovered a novel mechanism whereby elevated expression of miR-130a in t(8,21) AML endows enhanced self-renewal and perpetuates the aberrant molecular programs driven by AML1-ETO through inhibition of TBL1XR1.

Downregulation of GLI3 Expression Mediates Chemotherapy Resistance in Acute Myeloid Leukemia.

Aberrant activation of the hedgehog (HH) pathway is observed in many neoplasms, including acute myeloid leukemia (AML). The glioma-associated oncogene homolog (GLI) transcription factors are the main downstream effectors of the HH signaling cascade and are responsible for the proliferation and maintenance of leukemic stem cells, which support chemotherapy resistance and leukemia relapse. Cytarabine (Ara-C)-resistant variants of AML cell lines were established through long-term cultivation with successively increasing Ara-C concentrations. Subsequently, differences in GLI expression were analyzed by RT-qPCR. GLI3 mRNA levels were detectable in parental Kasumi-1, OCI-AML3, and OCI-AML5 cells, whereas GLI3 expression was completely silenced in all resistant counterparts. Therefore, we generated GLI3-knockdown cell lines using small hairpin RNAs (shRNA) and evaluated their sensitivity to Ara-C in vitro. The knockdown of GLI3 partly abolished the effect of Ara-C on colony formation and induction of apoptosis, indicating that GLI3 downregulation results in Ara-C resistance. Moreover, we analyzed the expression of several genes involved in Ara-C metabolism and transport. Knockdown of GLI3 resulted in the upregulation of SAM and HD domain-containing protein 1 (SAMHD1), cytidine deaminase (CDA), and ATP-binding cassette C11 (ABCC11)/multidrug resistance-associated protein 8 (MRP8), each of which has been identified as a predictive marker for Ara-C response in acute myeloid leukemia. Our results demonstrate that GLI3 downregulation is a potential mechanism to induce chemotherapy resistance in AML.

Cytological and spectroscopic characteristics of c-KIT N822K mutation in core binding factor acute myeloid leukemia cells.

The frequency of N822K mutation is high in the A-loop region of c-KIT which is highly associated with poor prognosis of core binding factor acute myeloid leukemia. The current work used common assays including cell cycle, apoptosis, clone formation and western blot to perform cytological detection for HL60 (wild type), NB4 (carrying t[15;17] chromosome translocation) and Kasumi-1 (with c-KIT N822K mutation); and meanwhile, the laser tweezers Raman spectroscopy (LTRS) was also used to perform label-free detection of single living cells. The results demonstrated that Kasumi-1 cell line bearing c-KIT N822K mutation has a stable cell cycle, while there was a significant difference between early and late apoptosis within 48 hours. The LTRS detection initially reflected the spectral differences induced by genetic abnormalities and highlighted progressive patterns of DNA and amino acids band contents which were appropriately consistent with that of cell clone ratio and the c-KIT phosphorylation level. It is concluded that methodology of LTRS-based single living cell characterization could be potential and effective to reveal gene mutation-induced cell differentiation.

DT-13 induced apoptosis and promoted differentiation of acute myeloid leukemia cells by activating AMPK-KLF2 pathway

Acute myeloid leukemia (AML) is a malignant disease originating from hematopoietic stem cells (HSC). Chemotherapy and/or HSC transplantation is unsatisfactory due to serious side effects, multidrug resistance, and high relapse rate. Thus, alternative strategies are urgently needed to develop more effective therapies. Liriope muscari baily saponins C (DT-13) is a novel compound isolated from Liriope muscari (Decne.) Baily, and exhibited a potent cytotoxicity against several solid tumors. However, the anti-AML activity of DT-13 and the potential mechanisms are still unknown. This study is the first to demonstrate that DT-13 had preferential cytotoxicity against AML cells, and remarkably inhibited proliferation and colony forming ability. Moreover, DT-13 induced the death receptor pathway-dependent apoptosis of HL-60 and Kasumi-1 cells by up-regulating Fas, FasL, DR5 and TRAIL as well as promoted the cleavage of caspase 8, caspase 3 and PARP. Meanwhile, DT-13 induced the differentiation with morphological change related to myeloid differentiation, elevated NBT and α-NAE positive cell rates, differentiation markers CD11b and CD14 as well as level of transcription factors C/EBPα and C/EBPβ. RNA-sequencing analysis revealed that KLF2 may be one of the potential targets regulated by DT-13. Further studies indicated that KLF2 played a critical role in DT-13-induced apoptosis and differentiation. Moreover, activation of AMPK-FOXO was proved to be the upstream of KLF2 pathway that contributed to the induction of apoptosis and differentiation by DT-13. Additionally, restoration of KLF2 by DT-13 was highly correlated with the AMPK-related histone acetylation mechanisms. Finally, DT-13 exhibited an obvious anti-AML effect in NOD/SCID mice with the engraftment of HL-60 cells. Our study suggests that DT-13 may serve as a novel agent for AML by AMPL-KLF2-mediated apoptosis and differentiation.

Upregulation of circ-0000745 in acute lymphoblastic leukemia enhanced cell proliferation by activating ERK pathway.

Acute lymphoblastic leukemia (ALL) is a common hematologic malignancy. Circular RNAs (circRNAs) have been reported as an important factor in regulating cellular process expressed in all hematopoietic compartment. But the molecular mechanism of circRNAs in ALL remain unclear. In this study, we observed circ-0000745 upregulated in leukemia cells (Kasumi-1 and KG-1). Upregulated the expression of circ-0000745 significantly enhanced the proliferation of Kasumi-1 and KG-1 cells, and reduced the ratio of apoptosis cells. Knock down the endogenous expression of circ-0000745 suppressed the cell proliferation and increased the ratio of apoptosis cells. Furthermore, western blot assay showed that overexpression of circ-0000745 increased the activity of ERK1/2. Altogether, these results revealed that upregulated circ-0000745 in leukemia cells could promoter cell viability by increasing the activation of ERK pathway. This study supported the important of circRNAs in the process of acute lymphoblastic leukemia, and provided a new biomarker on ALL diagnosis and therapy.

Human Cytomegalovirus Decreases Major Histocompatibility Complex Class II by Regulating Class II Transactivator Transcript Levels in a Myeloid Cell Line.

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that encodes many proteins to modulate the host immune response. Extensive efforts have led to the elucidation of multiple strategies employed by HCMV to effectively block NK cell targeting of virus-infected cells and the major histocompatibility complex (MHC) class I-primed CD8+ T cell response. However, viral regulation of the MHC class II-mediated CD4+ T cell response is understudied in endogenous MHC class II-expressing cells, largely because the popular cell culture systems utilized for studying HCMV do not endogenously express MHC class II. Of the many cell types infected by HCMV in the host, myeloid cells, such as monocytes, are of particular importance due to their role in latency and subsequent dissemination throughout the host. We investigated the impact of HCMV infection on MHC class II in Kasumi-3 cells, a myeloid-progenitor cell line that endogenously expresses the MHC class II gene, HLA-DR. We observed a significant reduction in the expression of surface and total HLA-DR at 72 h postinfection (hpi) and 120 hpi in infected cells. The decrease in HLA-DR expression was independent of the expression of previously described viral genes that regulate the MHC class II complex or the unique short (US) region of HCMV, a region expressing many immunomodulatory genes. The altered surface level of HLA-DR was not a result of increased endocytosis and degradation but was a result of a reduction in HLA-DR transcripts due to a decrease in the expression of the class II transactivator (CIITA).IMPORTANCE Human cytomegalovirus (HCMV) is an opportunistic herpesvirus that is asymptomatic for healthy individuals but that can lead to severe pathology in patients with congenital infections and immunosuppressed patients. Thus, it is important to understand the modulation of the immune response by HCMV, which is understudied in the context of endogenous MHC class II regulation. Using Kasumi-3 cells as a myeloid progenitor cell model endogenously expressing MHC class II (HLA-DR), this study shows that HCMV decreases the expression of HLA-DR in infected cells by reducing the transcription of HLA-DR transcripts early during infection independently of the expression of previously implicated genes. This is an important finding, as it highlights a mechanism of immune evasion utilized by HCMV to decrease the expression of MHC class II in a relevant cell system that endogenously expresses the MHC class II complex.

Kasumi leukemia cell lines: characterization of tumor genomes with ethnic origin and scales of genomic alterations

Kasumi-1 has played an important role in an experimental model with t(8;21) translocation, which is a representative example of leukemia cell lines. However, previous studies using Kasumi-1 show discrepancies in the genome profile. The wide use of leukemia cell lines is limited to lines that are well-characterized. The use of additional cell lines extends research to various types of leukemia, and to further explore leukemia pathogenesis, which can be achieved by uncovering the fundamental features of each cell line with accurate data. In this study, ten Kasumi cell lines established in Japan, including five that were previously unknown, have been characterized by SNP microarray and targeted sequencing. SNP genotyping suggested that the genetic ancestry in four of the ten Kasumi cell lines was not classified as Japanese but covered several different east-Asian ethnicities, suggesting that patients in Japan are genetically diverse. TP53 mutations were detected in two cell lines with complex array profiles, indicating chromosomal instability (CIN). A quantitative assessment of tumor genomes at the chromosomal level was newly introduced to reveal total DNA sizes and Scales of Genomic Alterations (SGA) for each cell line. Kasumi-1 and 6 derived from relapsed phases demonstrated high levels of SGA, implying that the level of SGA would reflect on the tumor progression and could serve as an index of CIN. Our results extend the leukemia cellular resources with an additional five cell lines and provide reference genome data with ethnic identities for the ten Kasumi cell lines.

Simultaneous Measurement of Tricarboxylic Acid Cycle Intermediates in Different Biological Matrices Using Liquid Chromatography-Tandem Mass Spectrometry; Quantitation and Comparison of TCA Cycle Intermediates in Human Serum, Plasma, Kasumi-1 Cell and Murine Liver Tissue.

The tricarboxylic acid (TCA) cycle is a central part of carbon and energy metabolism, also connecting to glycolysis, amino acid, and lipid metabolism. The quantitation of the TCA cycle intermediate within one method is lucrative due to the interest in central carbon metabolism profiling in cells and tissues. In addition, TCA cycle intermediates in serum have been discovered to correspond as biomarkers to various underlying pathological conditions. In this work, an Liquid Chromatography-Mass Spectrometry/Mass Spectrometry-based quantification method is developed and validated, which takes advantage of fast, specific, sensitive, and cost-efficient precipitation extraction. Chromatographic separation is achieved while using Atlantis dC18 2.1 mm × 100 mm, particle size 3-μm of Waters column with a gradient elution mobile phase while using formic acid in water (0.1% v/v) and acetonitrile. Linearity was clearly seen over a calibration range of: 6.25 to 6400 ng/mL (r2 > 0.980) for malic acid; 11.72 to 12,000 ng/mL (r2 > 0.980) for cis-aconitic acid and L-aspartic acid; 29.30 to 30,000 ng/mL (r2 > 0.980) for isocitric acid, l-serine, and l-glutamic acid; 122.07 to 125,000 ng/mL (r2 > 0.980) for citric acid, glycine, oxo-glutaric acid, l-alanine, and l-glutamine; 527.34 to 540,000 ng/mL (r2 > 0.980) for l-lactic acid; 976.56 to 1,000,000 ng/mL (r2 > 0.980) for d-glucose; 23.44 to 24,000 ng/mL (r2 > 0.980) for fumaric acid and succinic acid; and, 244.14 to 250,000 ng/mL (r2 > 0.980) for pyruvic acid. Validation was carried out, as per European Medicines Agency (EMA) “guidelines on bioanalytical method validation”, for linearity, precision, accuracy, limit of detection (LOD), limit of quantification (LLOQ), recovery, matrix effect, and stability. The recoveries from serum and tissue were 79–119% and 77–223%, respectively. Using this method, we measured TCA intermediates in serum, plasma (NIST 1950 SRM), and in mouse liver samples. The concentration found in NIST SRM 1950 (n = 6) of glycine (246.4 µmol/L), l-alanine (302.4 µmol/L), and serine (92.9 µmol/L).

Chidamide inhibits t(8;21) AML cell proliferation and AMK1/ETO and C-KIT expression by inhibiting ERK1/2 signaling pathway.

Backgroundt(8;21) acute myeloid leukemia (AML) is a highly heterogenous hematological malignancy. Histone deacetylases inhibitors (HDACi) are a group of small-molecule compounds with extensive anti-tumor activity. Chidamide (CS055) is a selective HDACi independently developed by China. We aimed to investigate its anti-tumor activity and mechanism in t(8;21) AML cells.MethodsHuman AML SKNO-1 cells were transfected with AML1/ETO-siRNA to obtain SKNO-1-si-A/E cells. Human acute monocytic leukemia U937 cells were transfected with AML1/ETO fusion gene to obtain U937-A/E cells. AML1/ETO-positive (Kasumi-1, U937-A/E and SKNO-1) and AML1/ETO-negative AML cells (HL-60, U937, SKNO-1-siA/E) were treated with chidamide (0.125, 0.25 and 0.5 µM). Cell proliferation was evaluated by CCK-8 assay. Cell apoptosis and cell cycle was detected by flow cytometry. Microarray profiling of SKNO-1 cells was performed. The level of histone 3 acetylation and ERK1/2 phosphorylation was determined by Western blot. AML1/ETO and C-KIT mRNA expression was detected by real time quantitative PCR (RT-qPCR). Kasumi-1 and SKNO-1 cells were treated with U0126, a special inhibitor of ERK1/2, and cell proliferation and ERK1/2 phosphorylation level was examined.ResultsChidamide inhibited the proliferation, induced cell cycle arrest, and stimulated cell apoptosis of AML1/ETO-positive AML cells. Microarray profiling showed that 13 differentially expressed genes were involved both in the “ERK1/2” pathway and “apoptosis” functions. Chidamide inhibited histone 3 acetylation and ERK1/2 phosphorylation in AML1/ETO-positive AML cells. U0126 inhibited the proliferation of AML1/ETO-positive AML cells via regulating the ERK1/2 pathway.ConclusionsOur results suggested that chidamide inhibits t(8;21) AML cell proliferation and AMK1/ETO and C-KIT expression by inhibiting ERK1/2 signaling pathway.

Dimethylation of eEF1A at Lysine 55 Plays a Key Role in the Regulation of eEF1A2 on Malignant Cell Functions of Acute Myeloid Leukemia.

Objective:This study aimed to explore whether eukaryotic translation elongation factor 1 alpha 2 affected cell proliferation, migration, and apoptosis via regulating the dimethylation of eukaryotic translation elongation factor 1 alpha at lysine 55 in acute myeloid leukemia.Methods:The expressions of eukaryotic translation elongation factor 1 alpha 2 and dimethylation of eukaryotic translation elongation factor 1 alpha at lysine 55 in acute myeloid leukemia cell lines and human normal bone marrow mononuclear cells (as control) were assessed. Control CRISPR-Cas9 lentivirus, eukaryotic translation elongation factor 1 alpha 2 knockout CRISPR-Cas9 lentivirus, vector plasmid, eukaryotic translation elongation factor 1 alpha 2 wild type overexpression plasmid, and eukaryotic translation elongation factor 1 alpha 2 with a K55R substitution overexpression plasmid were transfected into AML-193 and Kasumi-1 cells combined or alone, and were accordingly divided into 4 groups (Sgcontrol + vector group, SgeEF1A2 + vector group, SgeEF1A2 + eEF1A2WT group, and SgeEFIA2 + eEF1A2K55R group).Results:Eukaryotic translation elongation factor 1 alpha 2 and dimethylation of eukaryotic translation elongation factor 1 alpha at lysine 55 expressions were higher in AML-193, Kasumi-1, and KG-1 cell lines compared to the control. In AML-193 and Kasumi-1 cells, the knockout and compensated experiments revealed that eukaryotic translation elongation factor 1 alpha 2 promoted cell proliferation and migration but repressed apoptosis. Additionally, the knockout of eukaryotic translation elongation factor 1 alpha 2 decreased dimethylation of eukaryotic translation elongation factor 1 alpha at lysine 55 expression, meanwhile, eukaryotic translation elongation factor 1 alpha 2 wild type overexpression enhanced while eukaryotic translation elongation factor 1 alpha 2 with a K55R substitution overexpression did not influence the dimethylation of eukaryotic translation elongation factor 1 alpha at lysine 55 expression. Furthermore, eukaryotic translation elongation factor 1 alpha 2 wild type overexpression promoted cell proliferation, enhanced migration, and decreased apoptosis, but eukaryotic translation elongation factor 1 alpha 2 with a K55R substitution overexpression did not influence these cellular functions in AML-193 and Kasumi-1 cells, suggesting the implication of dimethylation of eukaryotic translation elongation factor 1 alpha at lysine 55 in eukaryotic translation elongation factor 1 alpha 2 mediated oncogenesis of acute myeloid leukemia.Conclusion:Eukaryotic translation elongation factor 1 alpha 2 and its dimethylated product may serve as therapeutic targets, and these findings may provide support for exploring novel strategies in acute myeloid leukemia treatment.

High Expression Level of SP1, CSF1R, and PAK1 Correlates with Sensitivity of Leukemia Cells to the Antibiotic Mithramycin

Acute myeloid leukemia (AML) is a genetically heterogeneous group of oncological diseases of the hematopoietic system, which are extremely difficult to treat. The development of new targeted drugs (Hylteritinib, Venetoclax) significantly improved the survival of patients, but resistance, as well as cytotoxic anti-leukemia drugs, often occurs. The search for new molecular targets for the development of effective approaches for the treatment of AML is very urgent. In blast cells of patients with AML, mutations, chromosomal rearrangements, and increased expression of a number of non-mutant genes, including transcription factor genes, are detected. The transcription factor Sp 1 binds to GC-rich regions of regulatory regions of various genes and thus controls their expression. Sp1 targets include genes responsible for proliferation, cell cycle regulation, and differentiation. In many malignant diseases, a high level of Sp1 gene expression is associated with an unfavorable prognosis, therefore, Sp1 is considered as a promising therapeutic target for cancer. In this paper, we estimated the expression levels of Sp1 in various malignant tissues. Increased Sp1 expression was detected in samples obtained from patients with AML, acute lymphoblastic leukemia, Ewing sarcoma, ovarian and kidney cancer. It is also shown that Sp1 expression correlates with the expression of genes encoding cytokine receptors and growth factors (CSF1R and IL6R), intracellular kinases (CSK, SYK, PAK1, ILK, JAK2), and transcription factor LMO2. The correlation between expression levels of Sp1 and CSF1R, SYK, Jak2 and LMO2 is also characteristic of transplanted human leukemia cells. We measured expression levels of Sp1, CSF1R, ILK, PAK1 in the cells of three transplantable lines of human leukemia and found increased levels of expression of these genes in Kasumi-1 cells. In addition, we showed that Kasumi-1 cells are most sensitive to Mitramycin, a drug that displaces Sp1 from its targets with DNA. Our data indicate the need to identify AML cells that are most sensitive to inhibition of Sp1 activity in order to assess the possibility of suppressing its activity in vivo.

PCDH17 functions as a common tumor suppressor gene in acute leukemia and its transcriptional downregulation is mediated primarily by aberrant histone acetylation, not DNA methylation.

We recently reported that methylation of PCDH17 gene is found in 30% of children with B-cell precursor acute lymphoblastic leukemia (ALL), and is significantly correlated to event-free or overall survival. We here evaluated PCDH17 mRNA expression in pediatric acute myeloid leukemia (AML) and ALL. PCDH17 mRNA expression levels in children with ALL/AML were lower than those in healthy counterparts. We next elucidated the mechanism underlying down-regulation of PCDH17 mRNA, using myeloid and lymphoid leukemic cell lines. Treatment with the histone deacetylase inhibitor trichostatin A (TSA) resulted in restoration of PCDH17 mRNA expression and growth inhibition in K562, HL60, REH, and RCH-ACV cell lines. Upregulation of PCDH17 mRNA expression resulted from histone H3 acetylation. Knockdown of the PCDH17 gene, caused by transduction of PCDH17-targeted shRNA, significantly enhanced the proliferation of KU812 cells. Meanwhile, overexpression of PCDH17 via retroviral-particle transfection substantially inhibited the growth of Kasumi1 cells. The fold-increase in PCDH17 mRNA expression mediated by 5-azacytidine, an inhibitor of DNA methyltransferase, was fundamentally lower than that produced by TSA. In conclusion, our results suggest that PCDH17 gene functions as a common tumor suppressor gene in leukemic cells, and that histone deacetylase inhibitors re-express PCDH17 mRNA to a greater extent than demethylation reagents.

Stable depletion of RUNX1-ETO in Kasumi-1 cells induces expression and enhanced proteolytic activity of Cathepsin G and Neutrophil Elastase.

The oncogenic fusion protein RUNX1-ETO is a product of the t(8;21) translocation and consists of the hematopoietic transcriptional master regulator RUNX1 and the repressor ETO. RUNX1-ETO is found in 10–15% of acute myeloid leukemia and interferes with the expression of genes that are essential for myeloid differentiation. The neutrophil serine protease Cathepsin G is one of the genes suppressed by RUNX1-ETO, but little is known about its impact on the regulation of other lysosomal proteases. By lentiviral transduction of the t(8;21) positive cell line Kasumi-1 with an RUNX1-ETO specific shRNA, we analyzed long-term effects of stable RUNX1-ETO silencing on cellular phenotypes and target gene expression. Stable anti RUNX1-ETO RNAi reduces both proliferation and apoptosis in Kasumi-1 cells. In addition, long-term knockdown of RUNX1-ETO leads to an upregulation of proteolytic activity in Kasumi-1 cells, which may be released in vitro upon cell lysis leading to massive degradation of cellular proteins. We therefore propose that protein expression data of RUNX1-ETO-silenced Kasumi-1 cells must be analyzed with caution, as cell lysis conditions can heavily influence the results of studies on protein expression. Next, a mass spectrometry-based approach was used to identify protease cleavage patterns in RUNX1-ETO-depleted Kasumi-1 cells and Neutrophil Elastase has been identified as a RUNX1-ETO candidate target. Finally, proteolytic activity of Neutrophil Elastase and Cathepsin G was functionally confirmed by si/shRNA-mediated knockdown in Kasumi-1 cells.

Ethyl acetate extract of Caesalpinia sappan L. inhibited acute myeloid leukemia via ROS-mediated apoptosis and differentiation

BackgroundThe dried heartwood of Caesalpinia sappan L. is traditionally prescribed in the formula of traditional Chinese medicine (TCM) for the treatment of acute myeloid leukemia (AML), while nothing is yet known of the active fractions and the underlying mechanisms. PurposeThis study aims to investigate the effect of the ethyl acetate extract of the dried heartwood of Caesalpinia sappan L. (C-A-E) on induction of apoptosis and promotion of differentiation in vitro and anti-AML activity in vivo. Study design/methodsThe aqueous extract was sequentially separated with solvents of increasing polarity and the active fraction was determined through the inhibition potency. The inhibition of the active fraction on cell viability, proliferation and colony formation was performed in different AML cells. Induction of apoptosis and the promotion of differentiation were further determined. Then, the level of the reactive oxygen species (ROS) and its potential role were assessed. Finally, anti-AML activity was evaluated in NOD/SCID mice. ResultsC-A-E exhibited the highest inhibition on the cell viability of HL-60 cells. Meanwhile, C-A-E significantly suppressed the proliferation and the colony formation ability of HL-60 and Kasumi-1 cells. Moreover, C-A-E significantly induced the apoptosis, which was partially reversed by Z-VAD-FMK. C-A-E also reduced the level of mitochondrial membrane potential, promoted the release of cytochrome C, decreased the Bcl-2/Bax ratio, and promoted the cleavage of caspase-9 and -3. In addition, Mdivi-1 (mitochondrial fission blocker) remarkably reduced the apoptosis caused by C-A-E. Meanwhile, C-A-E also induced the expression of Mff and Fis1 and increased the location of Drp1 in mitochondria. Furthermore, C-A-E obviously promoted the differentiation of AML cells characterized by the typic morphological changes, the increased NBT positive cells, as well as the increased CD11b and CD14 levels. Notably, C-A-E significantly enhanced the intracellular ROS level. Moreimportantly, C-A-E-mediated apoptosis and differentiation of HL-60 cells was significantly mitigated by NAC. Additionally, C-A-E also exhibited an obvious anti-AML effect in NOD/SCID mice with the injection of HL-60 cells. ConclusionsC-A-E exhibited an inhibitory effect on AML cells by inducing mitochondrial apoptosis and promoting differentiation, both of which were highly correlated to the activation of ROS.

LAG3 Inhibition Decreases AML-Induced Immunosuppression and Improves T Cell-Mediated Killing

Background: Acute Myeloid Leukemia (AML) is an aggressive hematologic malignancy with known immune dyregulation. In addition to their capacity to rapidly divide, AML cells directly inhibit the activation and proliferation of immune cells in culture. Immunosuppressive features observed in the bone marrow of AML patients include upregulation of Tregs and production of immunosuppressive cytokines (e.g., TGFβ). Irradiating AML cells diminishes their immunosuppressive capacity while maintaining antigen presentation, leading to increased activation of T cells in co-culture. We subsequently identified the immune checkpoint LAG3 as an important mediator of AML-induced immunosuppression and LAG3 modulation as potential treatment strategy. Methods: Normal PBMC were isolated from healthy donors. PBMC were co-cultured with non-irradiated and irradiated (40 Gy) human AML cell lines (Kasumi1 (K1), THP1) separately at a 1:2 ratio. On day 3 of co-culture, immunophenotypic characterization of T cells was performed on a flow cytometer using the following surface markers: CD3, CD4, CD8, CD25, CD137, CD154, PD-1, TIM3, TIGIT, and LAG3 and intracellular IFNg and FOXP3. Supernatant from co-culture media were analyzed for cytokine (IL-2, IL-6, IL-10 & TGFβ) secretion by ELISA. CFSE-labeled AML cells were incubated with healthy donor PBMCs in the presence or absence of LAG3, then viability was measured by 7-ADD on flow cytometry. PBMCs were also isolated from AML patients' peripheral blood and mononuclear cells were isolated from their respective bone marrow samples. Primary AML cultures were established in RPMI complete media with 20% FBS. CFSE-7-ADD killing assay was conducted after incubation of AML cells with autologous PBMCs. Results: Healthy donor PBMC co-cultured with irradiated K1 AML cells showed higher intracellular IFNg expression (11.8% ± 3.1 v. 7% ± 3.3; n=7, P=0.012) and higher CD137 expression (9.3% ± 1.21 v. 5.7% ± 3.4; n=7, P<0.001) on CD8+ T cells, and higher CD154 expression on CD4+ cells (44.7% ± 20.3 v. 26.3% ± 14.2; n=5, P=0.002) when compared to the non-irradiated K1-PBMC co-cultures. There were fewer Tregs (CD4+ CD25+ FOXP3+) in the PBMC co-cultured with irradiated K1 cells (1.96% ± 0.37 v. 3.39% ± 0.58; n=4, P=0.03) compared to the non-irradiated K1-PBMC co-cultures. LAG3 expression on CD8+ T cells co-cultured with irradiated K1 was decreased (11.8% ± 2.4 v. 17.5% ± 2.5; n=4, P=0.002) compared to the PBMC co-cultured with non-irradiated K1 cells. No other changes in checkpoint expression on CD8+ T cells were observed. No changes were observed in MHCI or PDL1 expression on non-irradiated K1 AML cells before or after co-culture with PBMC. We observed similar findings with healthy donor PBMC co-cultured with a different AML cell line, THP1; CD137 expression was higher on CD8+ T cells (17.6% v. 6.5%; P=0.02, n=3). ELISA of the supernatant of culture media showed higher mean OD for secreted TGFβ in the non-irradiated AML co-cultures compared to the irradiated AML co-cultures at 6 hours (2.5 v. 2.0, P=0.03, n=3) and 72 hours (7.9 v. 5.3, P=0.04, n=3). Adding anti-LAG3 antibody (3DS223H; 100 µg/ml) to PBMC co-cultured with non-irradiated AML cells resulted in higher IFNg (16.3% v. 6.6%, P=0.01, n=4) and CD137 expression (6.5% v. 4.1%, p=.007, n=4) on CD8+ cells and fewer Tregs (1.7% v. 3.8%, P=.04, n=4) compared to no antibody added. Healthy donor PBMC (n=3) were incubated with CFSE labeled AML cells (K1 and THP1) separately at an effector:target ratio of 5:1. The addition of anti-LAG3 antibody lead to increased killing of both K1 and THP1 AML cells at 4 and 24 hours (Figure 1A). To eliminate the HLA mismatch effect, we incubated PBMC from AML patients with autologous AML cells in the presence or absence of anti-LAG3 (Figure 1B). MHC-I blocking (W6/32, 30 µg/ml) lead to inhibition of cell mediated killing in the presence of anti-LAG3 (Figure 1B). Conclusion: In this in vitro model, AML cells showed immunosuppressive features with decreased activation of CD8+ T cells, upregulation of Tregs, increased secretion of TGFβ and higher expression of LAG3 on CD8+ T cells. Antibody blocking of LAG3 mitigated this effect, resulting in increased activation of T cells, fewer T regs and improved MHC-I-mediated killing against AML cells. These results demonstrate that the immunosuppressive effects of AML cells can be modulated through inhibition of LAG-3, suggesting a potential strategy for future combination therapy in AML. Disclosures Lin: Jazz Pharmaceuticals: Honoraria; Pfizer: Membership on an entity's Board of Directors or advisory committees.