Normal Bone Marrow Mononuclear Cells Research Articles

Mechanism and Clinical Significance of NAMPT in Multiple Myeloma

To investigate the mechanism and clinical value of nicotinamide phosphoribosyltransferase (NAMPT) in multiple myeloma (MM). RT-qPCR and Western blot were used to detect the expression of NAMPT in MM cells and normal bone marrow mononuclear cells. The biological function of NAMPT was analyzed by cell proliferation and apoptosis assay, small interfering RNA silencing, overexpression assay and chromatin immunoprecipitation assay. The mRNA and protein expression levels of NAMPT in MM cell lines (MM1R, MM1S, U266 and RPMI-8226) were significantly higher than those in normal bone marrow mononuclear cells (P < 0.001), and were most obvious in U266 cells. Compared with Si-NC group, the proliferation of U266 cells in Si-NAMPT group was significantly inhibited at 24, 48 and 72 h after transfection (P =0.006, P < 0.001, P =0.001), and the apoptosis rate of U266 cells was significantly increased at 48 h after transfection (P < 0.001). Compared with Flag-NC group, U266 cell proliferation in Flag-NAMPT group was significantly increased (P =0.003, P =0.002, P < 0.001), while the apoptosis rate decreased significantly at 48 h after transfection. The expression of NAMPT in U266 cells was regulated by XBP1 at transcriptional level. The proliferation rate of U266 cells with XBP1 or NAMPT stable knockout or MKC3946 pretreated with bortezomib was significantly decreased, the levels of BCL-2 mRNA and protein were also significantly decreased, while the levels of BAX mRNA and protein were significantly increased, moreover, the cleavage degree of caspase-3 significantly decreased, while caspase-3/7 activity increased dramatically (P < 0.05). The high expression of NAMPT in MM cell line can promote MM cell proliferation and inhibit apoptosis. NAMPT is regulated by IRE1α-XBP1 signaling pathway in U266 cells. Stable knockdown of NAMPT or blocking of IRE1α-XBP1 pathway can significantly increase the sensitivity of U266 cells to bortezomib.

Detection and identification of leukemic and normal cells using Raman spectroscopy and multivariate statistical analysis

Raman spectroscopy technique was successfully employed to study the difference between leukemic cells (Jurkat cell line) and normal human bone marrow mononuclear cells using near-infrared laser (785 nm) excitation in this study. Significant differences in Raman spectra from two kinds of cell lines showed special changes in the percentage of biochemical constituents in different cells. The obtained spectral data were used to develop diagnostic algorithms by multivariate statistical analysis, including principal component analysis combined with linear discriminate analysis. The multivariate statistical analysis method for classification between cancer and normal cells has achieved good differentiation with high diagnostic sensitivity (100%), specificity (100%), and accuracy (100%), respectively. These results showed that Raman spectroscopy is a novel, nondestructive method, that provided interesting information about the changes in biochemical properties of cells. Raman spectroscopy combined with multivariate statistical analysis can serve as a potential clinical diagnosis method of leukemia.

Targeting acute myeloid cell surface using a recombinant antibody isolated from whole-cell biopanning of a phage display human scFv antibody library.

To discover new therapeutic antibodies for treatment of acute myeloid leukemia (AML) without the requirement of a known antigen, a human single-chain variable fragment (scFv) library was used to isolate novel antibody fragments recognizing HL-60 AML cells. After three rounds of biopanning, scFv-expressing phages were selected to test for binding to the target cell by flow cytometry. The clone with highest binding specificity to HL-60 cells (designated y1HL63D6) was further investigated. Fluorescent staining indicated that y1HL63D6 scFv bound to a target located on the cell surface. Whole immunoglobulin, IgG-y1HL63D6 was then generated and tested for the binding against bone marrow mononuclear cells (BMMCs) from AML patients. Significantly higher fluorescent signals were observed for some patient samples when compared to normal BMMCs or non-AML patients' BMMCs. Next, the IgG-y1HL63D6 format was tested for antibody-dependent cell cytotoxicity (ADCC). The results demonstrated that IgG-y1HL63D6 but not the control antibody, trastuzumab, could mediate specific killing of HL-60 target cells. In conclusion, our results indicate that specific antibodies can be isolated by biopanning whole cells with a non-immunized human scFv antibody phage display library and that the isolated antibody against HL-60 cells showed therapeutic potential.

Effect of fetal liver condition media derived cytokines (IL-6 and Flt-3) on human bone marrow stem cells colony formation

Earlier research from our laboratory demonstrated the presence of stimulatory activity of different growth factors in the fetal liver (FL) extracts when collected in a medium known as fetal liver conditioned medium (FLCM) using Enzyme-linked Immunosorbent Assay (ELISA).In the present study, we have assessed two other cytokines viz. IL-6 and FMS like tyrosine kinase-3 (Flt-3) with the help of bioneutralization assay. FLCM was prepared by incubating fetal liver cells with Iscove’s Modified Dulbecco’s Medium (IMDM) containing 10% fetal bovine serum (FBS) and 10% Phytohemagglutinin and collected after 24hrs, 48hrs, 72 hrs. and on the 7th day of incubation. Clonal cultures were established for 1 X 105 normal bone marrow (BM) mononuclear cells (NBM MNC) per plate with methylcellulose medium containing cytokines SCF and EPO. Mean Colony forming units-granulocytes, erythrocytes, macrophages, megakaryocytes (CFU-GEMM) were assessed with and without the addition of FLCM. It was found that FLCM enhanced the number of colonies made by NBM MNCs. Further, cytokines IL-6 and Flt-3, present in FLCM, were bioneutralized with respective anti-cytokine antibodies. Neutralized FLCM was evaluated for the colony-forming potential of CFU-GEMM colonies. The maximum reduction of 42% was seen with 20 ng/ml of anti-IL-6 antibody. Maximum suppression up to 20% was observed with 0.7 ng/ml of anti Flt-3 antibody for CFU-GEMM colonies. Presence of cytokines IL-6 and Flt-3 in FL extracts and their colony stimulatory activity suggests that fetal liver infusion (FLI) may be a valuable alternative for managing BM recovery in certain clinical conditions such as AA.

APOBEC3B is preferentially expressed at the G2/M phase of cell cycle

APOBEC3B (A3B) is a cytosine deaminase that converts cytosine to uracil in single-stranded DNA. Cytosine-to-thymine and cytosine-to-guanine base substitution mutations in trinucleotide motifs (APOBEC mutational signatures) were found in various cancers including lymphoid hematological malignancies such as multiple myeloma and A3B has been shown to be an enzymatic source of mutations in those cancers. Although the importance of A3B is being increasingly recognized, it is unclear how A3B expression is regulated in cancer cells as well as normal cells. To answer these fundamental questions, we analyzed 1276 primary myeloma cells using single-cell RNA-sequencing (scRNA-seq) and found that A3B was preferentially expressed at the G2/M phase, in sharp contrast to the expression patterns of other APOBEC3 genes. Consistently, we demonstrated that A3B protein was preferentially expressed at the G2/M phase in myeloma cells by cell sorting. We also demonstrated that normal blood cells expressing A3B were also enriched in G2/M-phase cells by analyzing scRNA-seq data from 86,493 normal bone marrow mononuclear cells. Furthermore, we revealed that A3B was expressed mainly in plasma cells, CD10+ B cells and erythroid cells, but not in granulocyte-macrophage progenitors. A3B expression profiling in normal blood cells may contribute to understanding the defense mechanism of A3B against viruses, and partially explain the bias of APOBEC mutational signatures in lymphoid but not myeloid malignancies. This study identified the cells and cellular phase in which A3B is highly expressed, which may help reveal the mechanisms behind carcinogenesis and cancer heterogeneity, as well as the biological functions of A3B in normal blood cells.

Overexpression of chromodomain helicase DNA binding protein 5 (CHD5) inhibits cell proliferation and induces cell cycle arrest and apoptosis in chronic myeloid leukemia.

BackgroundChromodomain helicase DNA binding protein 5 (CHD5) was reported to be a tumor suppressor and our previous work showed CHD5 was epigenetically inactivated in human chronic myeloid leukemia (CML). This study aimed to investigate the effect of its overexpression on CML tumorigenesis.MethodsQuantitative reverse-transcriptase PCR and Western blotting analysis were used to detect the expression of CHD5 in human CML cell lines. The endogenous CHD5 expression was activated in two CML cell lines by CRISPR/dCas9-SAM system. In vitro cell function experiments were performed including proliferation, colony formation, apoptosis, autophagy, senescence and differentiation assays. Furthermore, tumorigenicity was evaluated in vivo in nude mice xenograft model.ResultsCHD5 was down-regulated in CML cell lines compare to normal bone marrow mononuclear cells (MCs). Cell proliferation after activating CHD5 was significantly inhibited. Moreover, overexpression of CHD5 induced G2/M phase arrest and apoptosis in CML cells. In a tumor xenograft mouse model, CHD5 restoration was found to sharply repress tumor growth. Compared with the control group, overexpression of CHD5 enhanced the expression of p21 and cdc2 phosphorylation, whereas decreased the protein level of Cyclin B1. Furthermore, experiments revealed that up-regulation of CHD5 activated caspase-3, while anti-apoptosis protein Bcl-2 expression was reduced in CML cells.ConclusionsCHD5 plays a role of anti-tumorigenic effects involved in CML cell proliferation, cell cycle arrest and apoptosis.

TNF-related apoptosis-inducing ligand (TRAIL) as a negative regulator of normal human erythropoiesis

AbstractThe impact of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) on normal hematopoietic development was investigated using adult peripheral blood CD34+ hematopoietic progenitor cells, induced to differentiate along the erythroid, megakaryocytic, granulocytic, and monocytic lineages by the addition of specific cytokine cocktails. TRAIL selectively reduced the number of erythroblasts, showing intermediate levels of glycophorin A (glycophorin Ainterm) surface expression, which appeared in liquid cultures supplemented with stem cell factor + interleukin 3 + erythropoietin at days 7-10. However, neither immature (day 4) glycophorin Adim erythroid cells nor mature (day 14) glycophorin Abright erythroblasts were sensitive to TRAIL-mediated apoptosis. Moreover, pre-exposure to TRAIL significantly decreased the number and size of erythroid colonies in semisolid assays. These adverse effects of TRAIL were selective for erythropoiesis, as TRAIL did not significantly influence the survival of cells differentiating along the megakaryocytic, granulocytic, or monocytic lineages. Furthermore, TRAIL was detected by Western blot analysis in lysates obtained from normal bone marrow mononuclear cells. These findings indicate that TRAIL acts in a lineage- and stage of differentiation-specific manner, as a negative regulator of normal erythropoiesis.

PPM1D Knockdown Suppresses Cell Proliferation, Promotes Cell Apoptosis, and Activates p38 MAPK/p53 Signaling Pathway in Acute Myeloid Leukemia.

Objectives:This study was to explore the effect of protein phosphatase, Mg2+/Mn2+ dependent 1D knockdown on proliferation and apoptosis as well as p38 MAPK/p53 signaling pathway in acute myeloid leukemia.Methods:The expression of protein phosphatase, Mg2+/Mn2+ dependent 1D was detected in acute myeloid leukemia cell lines including SKM-1, KG-1, AML-193, and THP-1 cells, and normal bone marrow mononuclear cells isolated from healthy donors. The knockdown of protein phosphatase, Mg2+/Mn2+ dependent 1D was conducted by transfecting small interfering RNA into AML-193 cells and KG-1 cells.Results:The relative messenger RNA/protein expressions of protein phosphatase, Mg2+/Mn2+ dependent 1D were higher in SKM-1, KG-1, AML-193, and THP-1 cells compared with control cells (normal bone marrow mononuclear cells). After transfecting protein phosphatase, Mg2+/Mn2+ dependent 1D small interfering RNA into AML-193 cells and KG-1 cells, both messenger RNA and protein expressions of protein phosphatase, Mg2+/Mn2+ dependent 1D were significantly reduced, indicating the successful transfection. Most importantly, knockdown of protein phosphatase, Mg2+/Mn2+ dependent 1D suppressed cell proliferation and promoted cell apoptosis in AML-193 cells and KG-1 cells. In addition, knockdown of protein phosphatase, Mg2+/Mn2+ dependent 1D enhanced the expressions of p-p38 and p53 in AML-193 cells and KG-1 cells. The above observation suggested that protein phosphatase, Mg2+/Mn2+ dependent 1D knockdown suppressed cell proliferation, promoted cell apoptosis, and activated p38 MAPK/p53 signaling pathway in acute myeloid leukemia cells.Conclusion:Protein phosphatase, Mg2+/Mn2+ dependent 1D is implicated in acute myeloid leukemia carcinogenesis, which illuminates its potential role as a treatment target for acute myeloid leukemia.

Long non-coding RNA GHET1/miR-105/RAP2B axis regulates the progression of acute myeloid leukemia.

Background: To explore the biological effects and potential molecular mechanisms of long non-coding RNA (lncRNA) gastric carcinoma proliferation enhancing transcript 1 (GHET1) in acute myeloid leukemia (AML).Methods: Fluorescence in situ hybridization was performed to determine the location of GHET1. Quantitative polymerase chain reaction (qPCR) was performed to verify RNA expression. GHET1 overexpression and knockdown were achieved by transfection of the expression vector or short hairpin RNA. Western blotting, qPCR, Cell Counting Kit-8 assay, JC-1 staining, and flow cytometry were performed to measure GHET1 function. The dual luciferase reporter assay was performed to confirm the relationship between microRNA 105 (mir-105) and Ras-related protein Rap-2B (RAP2B).Results: GHET1 was localized in the nucleus of NB4 cell lines. GHET1 expression was elevated in AML cell lines compared with normal bone marrow mononuclear cells. GHET1 knockdown led to inhibition of proliferation and promoted the differentiation and apoptosis of AML cell lines. Furthermore, GHET1 directly bound to miR-105 and downregulated miR-105 expression. MiR-105 overexpression suppressed proliferation and induced the differentiation and apoptosis of AML cell lines. In addition, RAP2B was confirmed to be a target gene of miR-105 and an inverse correlation was shown between their expression levels in AML cell lines; when miR-105 increased, Rap-2B level decreased and vice versa.Conclusion: This study demonstrated that the GHET1/miR-105/Rap2B axis may be a critical signaling pathway involved in AML progression.

Potential of circular RNA itchy E3 ubiquitin protein ligase as a biomarker and treatment target for multiple myeloma.

BackgroundThis study aimed to investigate the association of circular RNA itchy E3 ubiquitin protein ligase (circ-ITCH) expression with disease risk, clinical characteristics, progression-free survival (PFS) and overall survival (OS) of multiple myeloma (MM), and to explore the influence of circ-ITCH overexpression on MM cell activities in vitro.MethodsBone marrow samples from 92 MM patients and 30 healthy controls were collected, and circ-ITCH expression was detected by quantitative polymerase chain reaction. PFS and OS in MM patients were calculated. Circ-ITCH in human MM cell lines and normal bone marrow mononuclear cells (BMMCs) were detected. Circ-ITCH overexpression and control overexpression plasmids were transfected to U226 cell line, and cell proliferation as well as apoptosis were assessed.ResultsCirc-ITCH expression was under-expressed in MM patients compared to healthy controls. And receiver operating characteristic curve displayed that circ-ITCH could distinguish MM patients from healthy controls [area under curve: 0.809 (95% CI: 0.722–0.895)]. Additionally, circ-ITCH high expression was associated with decreased International Staging System (ISS) stage in MM patients. Kaplan-Meier curves and Cox’s regression analysis displayed that circ-ITCH expression was positively correlated with PFS and OS. In vitro, circ-ITCH expression was lower in MM cell lines (including RPMI8226, U226 and NCI-H929) compared to normal BMMCs. In U226 cells, cell proliferation was decreased but apoptosis was elevated by circ-ITCH overexpression.ConclusionsCirc-ITCH might serve as a potential biomarker and treatment target for MM.

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.

LncRNA MVIH knockdown inhibits the malignancy progression through downregulating miR-505 mediated HMGB1 and CCNE2 in acute myeloid leukemia

Background: This study aimed to investigate the regulatory role of long non-coding RNA associated with microvascular invasion in hepatocellular carcinoma ( lnc-MVIH ) in the progression of acute myeloid leukemia (AML) and the underlying mechanism. Methods: Lnc-MVIH expression was detected in AML cell lines AML-193, KG-1, HL-60, OCI-AML2 and primary normal bone marrow mononuclear cells (BMMC). The effect of lnc-MVIH knockdown on cell proliferation, apoptosis and miR-505 expression were detected by transfection of lnc-MVIH shRNA and control shRNA into KG-1 cells. And the effect of miR-505 knockdown on lnc-MVIH , cell proliferation, cell apoptosis as well as potential miR-505 target genes [ high mobility group box 1 ( HMGB1 ) and cyclin E2 ( CCNE2 )] in lnc-MVIH knockdown treated KG-1 cells was assessed by transfection of lnc-MVIH shRNA and lnc-MVIH shRNA & miR-505 shRNA into KG-1 cells. Results: Lnc-MVIH expression was elevated in AML-193, KG-1, OCI-AML2 cell lines, but similar in HL-60 cell line compared with primary normal BMMC. Lnc-MVIH knockdown inhibited cell proliferation but promoted cell apoptosis in KG-1 cells, meanwhile miR-505 expression was increased by lnc-MVIH knockdown in KG-1 cells. And in rescue experiments, miR-505 knockdown had no effect on expression of lnc-MVIH , while it increased the expressions of HMGB1 and CCNE2 , promoted cell proliferation, inhibited cell apoptosis in lnc-MVIH knockdown treated KG-1 cells. Conclusions: Lnc-MVIH knockdown inhibits cell proliferation but promotes cell apoptosis via regulating miR-505 mediated HMGB1 and CCNE2 in AML.

Identification of long non-coding RNA MVIH as a prognostic marker and therapeutic target in acute myeloid leukemia.

BackgroundThis study aimed to investigate the correlation of long non‐coding RNA microvascular invasion in hepatocellular carcinoma (lncRNA MVIH) with disease risk, disease conditions, and prognosis of acute myeloid leukemia (AML), and also to investigate the influence of lncRNA MVIH on AML cell activities in vitro.MethodsA total of 212 AML patients and 70 controls were consecutively recruited. Their bone marrow mononuclear cells (BMMCs) were isolated, and lncRNA MVIH was detected by reverse transcription quantitative‐polymerase chain reaction. In AML patients, complete remission (CR), event‐free survival (EFS), and overall survival (OS) were assessed. In vitro, lncRNA MVIH expression in AML cell lines was determined, and the effect of lncRNA MVIH on AML cell proliferation and apoptosis was assessed.ResultsLncRNA MVIH expression was increased in AML patients compared to controls, and receiver operating characteristic curve showed that lncRNA MVIH predicted elevated AML risk (area under curve: 0.742 [95% CI: 0.674‐0.810]). In AML patients, no correlation of lncRNA MVIH expression with French‐American‐British classification was observed, while lncRNA MVIH high expression correlated with worse risk stratification. Moreover, lncRNA MVIH expression negatively correlated with CR achievement, EFS and OS. In vitro, lncRNA MVIH was overexpressed in AML cell lines (KG‐1, ME‐1, and HT‐93) compared to normal BMMCs. Furthermore, lncRNA MVIH downregulation reduced KG‐1 cell proliferation but increased apoptosis, whereas lncRNA MVIH upregulation raised HL‐60 cell proliferation but decreased apoptosis.ConclusionLncRNA MVIH may not only serve as a prognostic marker but also act as a therapeutic target in AML.

LncRNA MVIH knockdown inhibits the malignancy progression through downregulating miR-505 mediated HMGB1 and CCNE2 in acute myeloid leukemia.

BackgroundThis study aimed to investigate the regulatory role of long non-coding RNA associated with microvascular invasion in hepatocellular carcinoma (lnc-MVIH) in the progression of acute myeloid leukemia (AML) and the underlying mechanism.MethodsLnc-MVIH expression was detected in AML cell lines AML-193, KG-1, HL-60, OCI-AML2 and primary normal bone marrow mononuclear cells (BMMC). The effect of lnc-MVIH knockdown on cell proliferation, apoptosis and miR-505 expression were detected by transfection of lnc-MVIH shRNA and control shRNA into KG-1 cells. And the effect of miR-505 knockdown on lnc-MVIH, cell proliferation, cell apoptosis as well as potential miR-505 target genes [high mobility group box 1 (HMGB1) and cyclin E2 (CCNE2)] in lnc-MVIH knockdown treated KG-1 cells was assessed by transfection of lnc-MVIH shRNA and lnc-MVIH shRNA & miR-505 shRNA into KG-1 cells.ResultsLnc-MVIH expression was elevated in AML-193, KG-1, OCI-AML2 cell lines, but similar in HL-60 cell line compared with primary normal BMMC. Lnc-MVIH knockdown inhibited cell proliferation but promoted cell apoptosis in KG-1 cells, meanwhile miR-505 expression was increased by lnc-MVIH knockdown in KG-1 cells. And in rescue experiments, miR-505 knockdown had no effect on expression of lnc-MVIH, while it increased the expressions of HMGB1 and CCNE2, promoted cell proliferation, inhibited cell apoptosis in lnc-MVIH knockdown treated KG-1 cells.ConclusionsLnc-MVIH knockdown inhibits cell proliferation but promotes cell apoptosis via regulating miR-505 mediated HMGB1 and CCNE2 in AML.

The implication of lncRNA expression pattern and potential function of lncRNA RP4-576H24.2 in acute myeloid leukemia.

BackgroundRecent studies have revealed that long noncoding RNAs (lncRNAs) may hold crucial triggers of the pathogenesis of hematological malignancies, while the studies evaluating the expression pattern of lncRNA in acute myeloid leukemia (AML) are few. Thus, this study aimed to investigate the implication of lncRNA expression pattern in AML development and progression.MethodsBone marrow samples from four AML patients and four controls were subjected to lncRNA sequencing. Then, bone marrow samples from 110 AML patients and 40 controls were proposed to real‐time quantitative polymerase chain reaction (RT‐qPCR) validation for 10 candidate lncRNAs. Clinical data and survival profiles were recorded in AML patients. Furthermore, lncRNA RP4‐576H24.2 expression in AML cell lines and its effect on AML cell proliferation and apoptosis were detected.ResultsLncRNA expression pattern by sequencing clearly distinguished AML patients from controls, and 630 upregulated and 621 downregulated lncRNAs were identified in AML patients compared to controls, which were mainly enriched in AML oncogene‐related biological process and pathways (such as neutrophil degranulation, leukocyte transendothelial migration, and hematopoietic cell lineage). RT‐qPCR validation observed that six lncRNAs correlated with AML risk, one lncRNA associated with risk stratification, and three lncRNAs correlated with survivals, among which lncRNA RP4‐576H24.2 was the only one correlated with AML susceptibility, risk stratification, and survivals. Further in vitro experiments showed that lncRNA RP4‐576H24.2 was upregulated in AML cell lines compared to normal bone marrow mononuclear cells (BMMCs), and promoted proliferation while inhibited apoptosis in HL‐60 and KG‐1 cells.ConclusionsLncRNA expression pattern is closely involved in the development and progression of AML, and several specific lncRNAs exhibit potential to be biomarkers for AML risk and prognosis. Besides, lncRNA RP4‐576H24.2 might be a potential oncogene in AML pathogenesis.

Induction of apoptosis and differentiation by Na/H exchanger 1 modulation in acute myeloid leukemia cells

We investigated the effect of the modulation of Na/H exchanger 1 (NHE1) on apoptosis, differentiation, and chemoresistance in acute myeloid leukemia (AML) cells to evaluate the possibility of NHE1 modulation as a novel therapeutic strategy for AML. The pHi of leukemia cell lines except KG1a was higher than that of normal bone marrow mononuclear cells (BM MNCs). Notably, in K562, cytarabine (AraC)-resistant OCI-AML2, and primary leukemia cells, pHi was significantly higher than that of normal BM MNCs. Western blotting and real-time quantitative PCR confirmed that the increased NHE1 expression was responsible for the higher pHi. Specifically, compared to CD34+CD38+ leukemia cells, the mean fluorescence intensity of NHE1 was significantly higher in CD34+CD38− leukemic stem cells. The out of range in pHi by treatment with an NHE inhibitor, the amiloride analogue 5-(N,N-hexamethylene) amiloride (HMA), or an NHE activator, phorbol 12-myristate 13-acetate (PMA), resulted in dose- and time-dependent inhibition of leukemia cell proliferation. PMA induced CD14+ differentiation of leukemia cells, whereas HMA induced cell cycle arrest at the G1 phase. HMA could induce apoptosis of leukemia cells even in AraC-resistant cells and showed an additive effect on apoptosis in AraC-sensitive cells. Our result revealed that AML cells prefer more alkalic intracellular moiety than normal BM MNCs following increased NHE1 expression and that NHE1 modulation can induce apoptosis and differentiation of AML cells. These findings imply that NHE1 is a potential target in cytotoxic or differentiation-induction treatment for AML.

Abstract 2050: Cell penetrating peptide, ST101, disrupts ATF5 regulation of anti-apoptotic Bcl-2 family proteins, resulting in induction of cancer cell death in vitro and tumor growth inhibition/regression in vivo

Abstract Anti-apoptotic B cell lymphoma 2 (Bcl-2) family proteins are frequently overexpressed across a variety of tumors, resulting in tumor cell survival and resistance to therapy. Inhibition of the expression or activity of these survival factors is an attractive approach for cancer therapy. Activating transcription factor 5 (ATF5) regulates gene transcription of anti-apoptotic Bcl-2 family proteins in neural progenitor cells and a wide range of human cancer cells. Here, we describe ST101, a rationally designed, synthetic, D-amino acid, cell penetrating peptide therapeutic designed to disrupt the protein-protein interactions driving ATF5-regulated gene transcription. Exposure of HL60 promyelocytic leukemia cells and MCF7 breast adenocarcinoma cells to low micromolar concentration of ST101 resulted in a decrease in MCL-1, BCL-2 and BIRC5 (Survivin) mRNA expression at 4 and 24 hrs post exposure. Further, exposure to ST101 resulted in a dose-dependent loss of viability across a panel of human cancer cells, including MCF7, HL60, U251 glioblastoma, A375 melanoma, DU145 prostate cancer, and A549 lung adenocarcinoma, characterized by an increase in annexin V and PI staining by flow cytometry peaking 48 hrs post exposure, resulting in a median half maximal effective concentration (EC50) value of 4.0 micromolar. In contrast, normal human peripheral blood mononuclear cells and bone marrow mononuclear cells were resistant to ST101-mediated cell death, with &amp;gt;80 micromolar EC50 values. In mouse xenograft experiments, 25mg/kg ST101 administered three times per week for three weeks resulted in significant tumor regression in MCF7 and U251 subcutaneous tumors as well as tumor growth delay in HL60 subcutaneous tumors. Tumor growth remained significantly inhibited weeks after the last treatment in the MCF7 and U251 models. In summary, ST101 selectively kills cancer cell lines by decreasing BCL-2 family gene expression, resulting in significant reductions in tumor growth in mouse models. Taken together, these data validate ST101 as a potent peptide therapeutic candidate for a variety of solid tumor and hematologic malignancies. Citation Format: Jim A. Rotolo, Rick Ramirez, Mark Koester, Siok Leong, Lila Ghamsari, Gene Merutka, Barry J. Kappel. Cell penetrating peptide, ST101, disrupts ATF5 regulation of anti-apoptotic Bcl-2 family proteins, resulting in induction of cancer cell death in vitro and tumor growth inhibition/regression in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2050.

Metformintreatment Overcomes ATRA-Resistance in Acute Promyelocytic Leukemia and Increases FOXO3A Expression

Introduction:FOXO3A is a transcription factor shown to be involved in all-trans retinoic acid (ATRA)-induced granulocytic differentiation and apoptosis in acute promyelocytic leukemia (APL). Its biological activity may be significantly enhanced upon metformin, raising the possibility that ATRA and Metformin may act synergistically; which could be useful to overcome ATRA resistance. Despite progress in APL treatment, approximately 10-15% of patients will relapse after treatment with ATRA and anthracyclines and frequently present with ATRA resistance. Relapsed patients respond well to arsenic trioxide (ATO) treatment, but the cost of ATO is a significant barrier in many countries. Aims: Here, we investigated the effects of in vitro treatment with ATRA plus metformin in APL cell lines and primary blasts, and quantified FOXO3A expression and correlated its levels with treatment outcome in a cohort of patients enrolled in the International Consortium on Acute Leukemia (ICAPL2006) study. Finally, we evaluated the effect of induced overexpression of FOXO3A gene in regards to cell viability and proliferation. Methods: Primary leukemic blasts from hCG-PMLRARα transgenic mice (TM; n=4) and APL patients (age, 25-47y; n=4) were treated with metformin alone (5mM/ 10mM) or in combination with ATRA (1µM) and evaluated for cell viability. In addition, 106 patients (age, 18-82y) with newly diagnosed APL enrolled in the ICAPL2006 study were included. As controls, eight samples of bone marrow mononuclear cells (BMMC) from healthy volunteers (age, 18-60y) were analyzed. To validate our data, FOXO3A transcript levels from an independent cohort was used (31 patients from Amazonia!, Probe n. 204131_s_at, and five normal BMMC samples included in the same databank). NB4/NB4R2 (ATRA-resistant) cell lines were transduced with FOXO3A or empty vector (control). After synchronization using double thymidine block, transduced cells were submitted to proliferation and cell cycle assays. For dose-response curves, cells were treated with graded concentrations of ATRA, ATO and metformin. For the apoptosis analysis, cells were treated with ATRA (1µM), metformin (5mM) and combination for 24, 48 and 72 hours. The granulocytic differentiation in response to ATRA treatment was evaluated based on the CD11b surface levels. Results: In primary cells (from TM/APL patients) a 2-fold reduction in viable cells was observed after metformin and metformin plus ATRA treatment (P Disclosures Tallman:BioSight: Other: Advisory board; Daiichi-Sankyo: Other: Advisory board; ADC Therapeutics: Research Funding; AROG: Research Funding; AbbVie: Research Funding; Cellerant: Research Funding; Orsenix: Other: Advisory board. Ganser:Novartis: Membership on an entity9s Board of Directors or advisory committees. Lowenberg:Editorial Board "European Oncology & Haematology": Membership on an entity9s Board of Directors or advisory committees; Royal Academy of Sciences and Arts, The Netherlands: Membership on an entity9s Board of Directors or advisory committees; Supervisory Board, National Comprehensive Cancer Center (IKNL), Netherlands: Membership on an entity9s Board of Directors or advisory committees; Clear Creek Bio Ltd: Consultancy, Honoraria; international Scientific Advisory Board, Institute Gustave Roussy, Paris: Membership on an entity9s Board of Directors or advisory committees; Chairman Scientific Committee and Member Executive Committee, European School of Hematology (ESH, Paris, France): Membership on an entity9s Board of Directors or advisory committees; Elected member, Royal Academy of Sciences and Arts, The Netherlands: Membership on an entity9s Board of Directors or advisory committees; Editorial Board "The Netherlands Journal of Medicine": Membership on an entity9s Board of Directors or advisory committees; Editorial Board "International Journal of Hematology": Membership on an entity9s Board of Directors or advisory committees; "Up-to-Date", section editor leukemia: Membership on an entity9s Board of Directors or advisory committees; Agios Pharmaceuticals: Membership on an entity9s Board of Directors or advisory committees; Astex: Consultancy; Chairman, Leukemia Cooperative Trial Group HOVON (Netherlands): Membership on an entity9s Board of Directors or advisory committees; Celgene: Membership on an entity9s Board of Directors or advisory committees.

Leukemia cells detection based on electroporation assisted surface-enhanced Raman scattering

In this study, an electroporation-based surface-enhanced Raman scattering (SERS) technique was employed to differentiate the human myeloid leukemia cells from the normal human bone marrow mononuclear cells with the aim to develop a fast and label-free method for leukemia cell screening. The Ag nanoparticles were delivered into living cells by electroporation, and then high quality SERS spectra were successfully obtained from 60 acute promyelocytic leukemia cells (HL60 cell line), 60 chronic myelogenous leukemia cells (K562 cell line) and 60 normal human bone marrow mononuclear cells (BMC). Principal component analysis (PCA) combined with linear discriminant analysis (LDA) differentiated the leukemia cell SERS spectra (HL60 plus K562) from normal cell SERS spectra (BMC) with high sensitivity (98.3%) and specificity (98.3%). Furthermore, partial least squares (PLS) approach was employed to develop a diagnostic model. The model successfully predicted the unidentified subjects with a diagnostic accuracy of 96.7%. This exploratory work demonstrates that the electroporation-based SERS technique combined with PCA-LDA and PLS diagnostic algorithms possesses great promise for cancer cell screening.

Dual inhibition of Fes and Flt3 tyrosine kinases potently inhibits Flt3-ITD+ AML cell growth.

Acute myelogenous leukemia (AML) is often associated with activating mutations in the receptor tyrosine kinase, Flt3, including internal tandem duplications (ITDs) within the regulatory juxtamembrane region. Previous studies have linked Flt3-ITD to the activation of the Fes protein tyrosine kinase in AML, and RNAi-knockdown studies suggest that Fes may be required for Flt3 function. In this study, we tested Fes inhibitors from three different chemical classes for their growth-suppressive activity against Flt3-ITD+ myeloid leukemia cell lines (MV4-11, MOLM-13 and MOLM-14) vs. myeloid cells with wild-type Flt3 (THP-1). All Fes inhibitors selectively inhibited the growth of Flt3-ITD+ AML cells, with IC50 values for diaminopyrimidine and pyrrolopyridine inhibitors ranging from 19 to 166 nM. In contrast, a pyrazolopyrimidine inhibitor was less potent in Flt3-ITD+ AML cells, with IC50 values in the 1.0 μM range. In vitro kinase assays showed that the most potent inhibitors of Flt3-ITD+ AML cell proliferation blocked both Fes and Flt3-ITD kinase activity, while the pyrazolopyrimidine was more selective for Fes vs. Flt3-ITD. All three inhibitors induced significant apoptosis in Flt3-ITD+ AML cells, with potency equivalent to or greater than the established Flt3-ITD inhibitor, tandutinib. Transformation of TF-1 cells with Flt3-ITD resulted in constitutive activation of endogenous Fes, and rendered the cells highly sensitive to all three Fes inhibitors with IC50 values in the 30–500 nM range. The pyrrolopyridine compound also induced apoptotic responses in patient-derived Flt3-ITD+ AML bone marrow cells but not in normal bone marrow mononuclear cells. These results demonstrate that Fes kinase activity contributes to Flt3-ITD signaling in AML, and suggests that dual inhibition of both Flt3 and Fes may provide a therapeutic advantage for the treatment of Flt3-ITD+ AML.