Lymphocytic Leukemia Cell Line Research Articles

Phosphatidylinositol 3-kinase δ blockade increases genomic instability in B cells.

Activation-induced cytidine deaminase (AID) is a B-cell specific enzyme that targets immunoglobulin (Ig) genes to initiate class switch recombination (CSR) and somatic hypermutation (SHM)1. Through off-target activity, however, AID has a much broader impact on genomic instability by initiating oncogenic chromosomal translocations and mutations involved in lymphoma development and progression2. AID expression is tightly regulated in B cells and its overexpression leads to enhanced genomic instability and lymphoma formation3. The phosphatidylinositol 3-kinase (PI3K) δ pathway plays a key role in AID regulation by suppressing its expression in B cells4. Novel drugs for leukemia or lymphoma therapy such as idelalisib, duvelisib or ibrutinib block PI3Kδ activity directly or indirectly5–8, potentially affecting AID expression and, consequently, genomic stability in B cells. Here we show that treatment of primary mouse B cells with idelalisib or duvelisib, and to a lesser extent ibrutinib, enhanced the expression of AID and increased somatic hypermutation (SHM) and chromosomal translocation frequency to the Igh locus and to several AID off-target sites. Both these effects were completely abrogated in AID deficient B cells. PI3Kδ inhibitors or ibrutinib increased the formation of AID-dependent tumors in pristane-treated mice. Consistently, PI3Kδ inhibitors enhanced AID expression and translocation frequency to IgH and AID off-target sites in human chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) cell lines, and patients treated with idelalisib, but not ibrutinib, showed increased SHM in AID off-targets. In summary, we show that PI3Kδ or BTK inhibitors increase genomic instability in normal and neoplastic B cells by an AID-dependent mechanism, an effect that should be carefully considered as such inhibitors are administered for years to patients.

Yessotoxin, a Marine Toxin, Exhibits Anti-Allergic and Anti-Tumoural Activities Inhibiting Melanoma Tumour Growth in a Preclinical Model.

Yessotoxins (YTXs) are a group of marine toxins produced by the dinoflagellates Protoceratium reticulatum, Lingulodinium polyedrum and Gonyaulax spinifera. They may have medical interest due to their potential role as anti-allergic but also anti-cancer compounds. However, their biological activities remain poorly characterized. Here, we show that the small molecular compound YTX causes a slight but significant reduction of the ability of mast cells to degranulate. Strikingly, further examination revealed that YTX had a marked and selective cytotoxicity for the RBL-2H3 mast cell line inducing apoptosis, while primary bone marrow derived mast cells were highly resistant. In addition, YTX exhibited strong cytotoxicity against the human B-chronic lymphocytic leukaemia cell line MEC1 and the murine melanoma cell line B16F10. To analyse the potential role of YTX as an anti-cancer drug in vivo we used the well-established B16F10 melanoma preclinical mouse model. Our results demonstrate that a few local application of YTX around established tumours dramatically diminished tumour growth in the absence of any significant toxicity as determined by the absence of weight loss and haematological alterations. Our data support that YTX may have a minor role as an anti-allergic drug, but reveals an important potential for its use as an anti-cancer drug.

CXCR3 Upregulation Effect of T Lymphocytic Leukemia Cell Invasion in Vitro

ObjectiveThe study was to explore chemokine receptor CXCR3 influence on invasion of T lymphocytic leukemia cell.MethodsMouse CXCR3 gene was amplified from T vector containing mouse CXCR3 by PCR, and constructed the mouse CXCR3 overexpressing lentivirus carrying GFP & Puromycin (puro). Jurkat cell line was infected by the constructed CXCR3 overexpression lentivirus. Puromycin was added to screene the successfully infected lymphoblastic leukemia cells. CXCR3 expression on Jurkat cells was analyzed by flow cytometre. Constructed CXCR3-LV5-Jurkat cells were added into the Transwell upper chamber covered with Matrigel. Ligand of CXCR3(CXCL10) was added to the lower chamber. Cell number in the upper chamber and lower chamber which used to calculate the invasion rate was counted by flow cytometry respectively.ResultsGFP+ Jurkat cells was observed less than <10% after lentivirus infection 96h. GFP+Jurkat cells achieved to 90% by Puromycin selection. The expression of CXCR3 on CXCR3-LV5-Jurkat cell group was 90% higher than LV5-Jurkat cell group. CXCR3-LV5-Jurkat cell line stably overexpressed CXCR3 was successfully constructed. The invasion rate of CXCR3-LV5-Jurkat cell lines was significantly higher than LV5-Jurkat control group (12.71 %± 1.03% vs 6.82% ± 0.49%, p <0.0001).ConclusionOverexpression of CXCR3 on T lymphocytic leukemia cell line (Jurkat cell) can enhance the invasion of leukemia cells in vitro, CXCR3/CXCL10 path way may induce the T lymphoblastic leukemic cell invading the central nervous system. DisclosuresNo relevant conflicts of interest to declare.

A CD47xCD19 Bispecific Antibody That Remodels the Tumor Microenvironment for Improved Killing and Provokes a Memory Immune Response to Cancer B Cells

Up-regulation of CD47 in hematological and solid cancers correlates with poor clinical prognosis. CD47 interaction with SIRPα provides a ‘don’t eat me' signal that allows healthy cells to limit elimination by immune cells, in particular macrophages. Although tumor-associated macrophages (TAMs) are often considered pro-tumorigenic, several studies report a high phagocytic potential and tumoricidal function in the presence of therapeutic antibodies (Ab). Therefore, targeting the CD47-SIRPα pathway in the tumor microenvironment is an attractive approach to maximize the tumor killing potential of TAMs to boost tumor destruction. However, clinical development of monoclonal Abs to CD47 is likely to be hindered by the ubiquitous expression of CD47 leading to rapid drug elimination and toxicity including anemia. To address these concerns, we have created NI-1701, a bispecific Ab that drives efficacious binding only to CD19+B cells by pairing a high affinity anti-CD19 targeting arm to an anti-CD47 arm of optimized affinity..In addition to in vitro data demonstrating that the bispecific Ab, NI-1701, effectively kills CD19+ human tumor B cells through ADCP (antibody-dependent cellular phagocytosis) and antibody-dependent cell-mediated cytotoxicity (ADCC), we have observed significant tumor killing in vivo, as either a monotherapy or in a combination approach. NI-1701 controlled sub-cutaneously implanted Raji cell tumor growth in NOD/SCID mice in a manner dependent on the co-ligation of both CD19 and CD47. Examination of the excised tumors revealed that NI-1701 reshaped the tumor microenvironment by enhancing the tumoricidal activity of macrophages (i.e., more macrophages engulfing tumor cells), by promoting an antitumor M1-like phenotype, and reducing the proportion of CD11b+Gr1+myeloid-derived suppressor cells (MDSCs). Extending these findings to a disseminated in vivo model, NI-1701 eliminated tumor cells from the peripheral blood, bone marrow and liver in mice transplanted either with the B-Acute Lymphocytic Leukemia (B-ALL) cell line NALM-6 or with primary cells from B-ALL patients. Furthermore, NI-1701 also abrogated tumor growth more efficiently than the BTK inhibitor ibrutinib in a Diffuse Large B-Cell Lymphoma (DLBCL) patient-derived xenograft (PDX) mouse model.As combination therapies are gaining traction as successful treatment strategies in the clinic, we next tested the effect of blocking CD47 biology in combination with clinically validated molecules. Interestingly, in NOD/SCID mice implanted with Raji cells, NI-1701 was shown to be more efficacious at controlling tumor cell growth than Rituximab. A combination of NI-1701 and Rituximab was shown to act synergistically at controlling tumor growth and leading to tumor regression in some mice. Finally, in a syngeneic re-challenge model, using bispecific reagents targeting CD47 blockade to the A20 murine B-cell lymphoma, we observed the induction of a durable and protective anti-tumor response when combined with a single administration of cyclophosphamide.Importantly, in vitro safety studies demonstrate a favorable binding profile of NI-1701 to B cells compared with erythrocytes, no evidence of platelet activation or aggregation and no haemagglutination at and above anticipated therapeutic concentrations. Single and multiple dose studies in non-human primates demonstrated favorable elimination kinetics and no effects on hematological parameters (e.g., red blood cell and platelet counts) up to 100mg/kg, the highest dose tested.Taken together, we describe a novel bispecific approach that balances a safe yet effective blockade of CD47 with a high selectivity for a B cell associated antigen resulting in impressive tumor cell killing in a range of preclinical models. The effects on both the reshaping of the tumor microenvironment and the induction of long term tumor immunity provide further evidence that manipulation of myeloid lineage cells (e.g., macrophages and dendritic cells) is a promising approach for the next frontier in immune-oncology treatment strategies. NI-1701 is in preclinical enabling studies in preparation for a Phase I clinical study in patients with CD19+ B cell malignancies, planned for early 2017. DisclosuresFerlin:Novimmune S.A.: Employment, Equity Ownership. Chauchet:Novimmune S.A.: Employment. Buatois:Novimmune S.A.: Employment. Salgado-Pires:Novimmune S.A.: Employment. Shang:Novimmune S.A.: Employment. Dheilly:Novimmune S.A.: Employment. Masternak:Novimmune S.A.: Employment. Johnson:Novimmune S.A.: Employment. DiPersio:Incyte Corporation: Research Funding. Kosco-Vilbois:Novimmune S.A.: Employment. Fischer:Novimmune S.A.: Employment.

JQ1, a Potential Therapeutic Molecule for Myeloid Leukemia with PTEN Deficiency

Acute myeloid leukemia (AML) is a hematologic malignancy that continues to have high relapse and treatment-related mortality rates, despite recent advances in clinical management and therapy. Janus kinase (JAK) inhibitors inhibit the activity of the JAK/STAT pathway and have demonstrated some clinical responses in AML patients. However, survival analysis suggests that more than half of AML patients do not benefit from treatment with JAK inhibitors. Furthermore, PTEN deficiency is frequently found in patients in the late stages of cancer, which causes hyperactivated AKT and MAPK pathways. However, emerging data suggests that leukemia cells with PTEN deficiency are resistant to MAPK inhibitors. Over the past decade, it has been demonstrated that dysregulated epigenetics play an important role in myeloid leukemogenesis. The bromodomain and extraterminal domain (BET) family includes adaptor proteins Brd2, Brd3, Brd4, and Brdt that regulate gene expression via binding to acetylated chromatin and subsequently activating RNA Polymerase II driven transcriptional elongation, resulting in the promotion of gene expression. BRD4 is a BET protein required for disease maintenance in AML. JQ1 is a small molecule that interferes with transcriptional regulators, such as BRD4, by preventing them from interacting with acetylated regions of the genome and thus inhibiting the transcriptional activation of BRD4 target genes. Prior research in lymphocytic leukemia cell lines suggests that JQ1 also decreases STAT5-dependent gene transcriptional activities. We hypothesize that the inhibition of BET proteins may correct the over-activated transcriptional activities in myeloid leukemia cells and induce disease regression. We tested our hypothesis in PTEN deficient myeloid leukemia cell lines, TF-1a and K562, and used human cord blood mononuclear cells (CB) for normal cell comparison. Methods: 1) To test whether JQ1 can inhibit colony formation, we seeded cells on 0.3% agar and McCoys’ 5A medium supplemented with nutrients and 15% fetal bovine serum, without cytokines, and added JQ1 diluents to the cultures at concentrations of 32.5-1000nM overnight after the cultures were established. 2) To test whether JQ1 can inhibit leukemia cell proliferation, we cultured cells in liquid medium with JQ1 for 48-72 hours, and quantified the viable cells using alamarBlue® assay. 3) To investigate whether JAK/STAT5 activity is altered by JQ1 in leukemia cells, we quantified phosphorylated STAT5 (pSTAT5) in cells via flow cytometry and western blot. We treated the cells with JQ1 at various concentrations for 2 hours and then stimulated the cells for 15 minutes in medium with 0.5% BSA and 10ng/mL GM-CSF prior to staining the cells with anti-pStat5 (pY694) antibody conjugated with Alexa Fluor® 647 for FACS analysis or lysing the cells for western blot analysis. Results: In the colony formation assay, we found that TF-1a cells were more sensitive to JQ1 than the CB cells and K562, with an IC50 of 62.5-125 nM for TF-1a cells (p<0.0001), and 250-500nM for both CB and K562 cells, respectively. Proliferation assay results also supported that TF1a cells are sensitive to JQ1 with an IC50 of 125-250nM, whereas neither CB nor K562 reached the IC50 in the tested concentration range. This suggests that the IC50 of JQ1 for TF1a cells is achievable at concentrations that are mostly nontoxic to normal CB cells, but K562 cells are not sensitive to JQ1. FACS analysis revealed that pSTAT5 is constitutively activated in K562 cells but not in TF-1a cells. Interestingly, the levels of pSTAT5 in both TF-1a and K562 cells were not altered by JQ1 treatment at tested concentrations, which was confirmed by western blot. Conclusions: Our data suggest: 1) JQ1 and other bromodomain inhibitors could be potential therapeutic molecules for selected myeloid leukemias; 2) JQ1 inhibition on colony formation and proliferation in TF-1a cells is not pSTAT5 related. Further studies are underway to test whether JQ1 is effective in primary mouse leukemia cells with Pten deficiency. DisclosuresNo relevant conflicts of interest to declare.

29P - Anticancer activity of the mTOR inhibitor (everolimus) and dual mTORC1/mTORC2 Inhibitor (AZD2014) on mouse lymphocytic leukemia both in vitro and in vivo

The mammalian target of rapamycin (mTOR) controls cell growth and enlargement and has been found to be aberrant in a wide variety of malignancies. Allosteric mTOR inhibitors, which inhibit mTORC1 but not mTORC2, result in feedback activation of AKT signaling, which can attenuate their antitumor activity. AZD2014 is a second generation mTOR inhibitor that blocks activation of both mTORC1 and mTORC2, and activates apoptosis in cancer cells without activation of AKT signaling. Here, we investigated the therapeutic efficacy of everolimus and AZD2014 in lymphocytic leukemia cell line (L1210) and mouse xenograft model. Cytotoxicity effect of AZD2014 and everolimus in L1210 cells was assessed after 24 and 48 h by using cell viability assays, clonogenic survival assays, and cell cycle analyses. Cell cycle, mTOR signal transduction pathway and the relative regulatory molecules were examined in mTOR inhibitor-treated L1210 cells by western blotting to detect protein expression. Then, the in vivo anti-leukemic effect of AZD2014 was assessed in L1210 cell-transplanted DBA/2 mice. AZD2014 significantly inhibited L1210 cell proliferation with an IC50 of 100nM. In contrast, everolimus was a poor growth inhibitor of L1210 cells (IC50 > 200nM). Treatment with AZD2014 was more effective than RAD001 to down-regulate the levels of mTORC1 downstream effectors, including S6K1, 4EBP1,eIF4E and a significant decrease in protein levels of rictor, a component of mTORC2 protein.We also observed inhibition of mTORincreased G1 arrest by reducing cyclin D1 and CDK4 levels, which augmented growth-inhibitory effect of L1210 cells. In vivo, AZD2014 oral administration significantly inhibited the growth of L1210 cell xenograft in DBA/2 mice, and the mice survival was dramatically improved. These data indicate that AZD2014 may be a better therapeutic agent than mTORC1 inhibitors to enhance the antitumor activity of lymphocytic leukemia both in vitro and under xenograft model in vivo conditions. Antitumor activity of AZD2014 was inhibited of both mTORC1 and mTORC2 activity and cell cycle arrest, leading to lymphocytic leukemia growth inhibition.

Abstract A16: Establishment and characterization of CCGL007, a novel human chronic lymphocytic leukemia cell line

Abstract Chronic lymphocytic leukemia (CLL) is a monoclonal disorder characterized by a progressive accumulation of functionally incompetent lymphocytes. However, lack of immortalized cell line representative of the CLL disease has hampered a full understanding of disease pathogenesis and development of novel therapeutic agents for treatment. There were very few representative CLL cell lines are available now. To this end, we have tested different culture condition with addition of growth factor supplements to generate CLL cell lines from highly purified peripheral blood B cells of CLL patients. Recently, we have successfully established a new CLL cell line, named CCGL007. CCGL007 cells grow as suspension in liquid culture. Surface marker analysis with a panel of monoclonal antibodies revealed that CCGL007 cell line expresses B cell markers. The chromosomal, immunophenotypic, molecular biologic characteristics, and in vivo tumorigenesis potential of CCGL007 is under investigation. We have used this cell model to test the efficacy of selected standard chemotherapy drugs and new therapeutic agents, such as ibrutinib and several novel in-house BTK inhibitors, against CLL cell growth, and CCGL007 represents as a suitable preclinical model for testing pharmacological agents. Citation Format: Ching-Chuan Kuo, Chih-Cheng Chen, Chih-Hsiang Huang, Lin Li-Mei. Establishment and characterization of CCGL007, a novel human chronic lymphocytic leukemia cell line. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr A16.

Effect of BRD4 Inhibitor JQ1 on Proliferation Inhibition and Apotosis Induction in Jurkat Cells

To investigate the effect and possible mechanism of bromo-domain inhibitors (JQ1) on proliferation inhibition and inducing apoptosis of acute T lymphocyte leukemia cell line (Jurkat) . Jurkat cell line was treated by JQ1 at different concentrations. MTT was used to detect the cell proliferation inhibition rate. The flow cytometry with AnnexinV-FITC/PI fluorescence staining was used to detect the changes of apoptosis rate, and real-time fluorescent quantitative PCR was used to detect c-Myc/Notch1 gene expression levels. With the increasing of drug concentration and prolonging of time, the inhibitory rate of Jurkat cell growth was enhanced in time-dose dependent manner; Jurkat cells was treated by 0.8, 1.6, and 4 µ mol/LJQ1 for 48 h and 72 h, the cell apoptosis rate was enhanced with the increase of drug concentration and prolonging of time, and the difference was statistically different in comparison with the control group(P<0.05); PCR detection indicated that Notch1 and c-Myc mRNA expression was reduced in 48 h after JQ1 treatment, which was statistically different from the control group,(P<0.05) . JQ1 can effectively inhibit the growth of Jurkat cell line, and potentially induce apoptosis through Notch1 and c-Myc gene. Hence JQ1 may be one of new methods used to treat T-ALL.

Different expression levels of glycans on leukemic cells-a novel screening method with molecularly imprinted polymers (MIP) targeting sialic acid.

Sialic acid (SA) is normally expressed on the cell membranes and is located at the terminal position of the sugar chains. SA plays an important role for regulation of the innate immunity, function as markers of the cells and can be recognized by a variety of receptors. Interestingly, the level of SA expression is increased on metastatic cancer cells. The availability of specific antibodies against SA is limited and, therefore, biomarker tools for detection of SA are lacking. We have recently presented a novel method for specific fluorescence labeling of SA molecular imprinted polymers (MIP). Here, we have performed an extended screening of SA expression by using SA-MIP and included four different chronic lymphocytic leukemia (CLL) cell lines, conveniently analyzed by flow cytometry and fluorescence microscopy. SA expression was detected in four cell lines at different levels, and the SA expression were verified with lectin-FITC. These results show that SA-MIP can be used as a plastic antibody for detection of SA using both flow cytometry and fluorescence microscopy. We suggest that SA-MIP can be used for screening of different tumor cells of various stages, including CLL cells.

Abstract 268: PARP3 inhibitors in cancer therapy

Abstract Poly-ADP-ribose-polymerase (PARP1) is an important regulator of DNA damage response (DDR). After induction of certain types of DNA damage, including nicks and double strand breaks (DSB)s, PARP1 is rapidly recruited to altered DNA sites were its catalytic activity produces protein-conjugated long-branched-poly-ADP-ribose (PAR) chains. These protein modifications result in the recruitment and activation of multiple proteins involved in DNA repair. Although there is considerable research describing the characteristics and activity of PARP1/2, there are 15 other PARP family members that are less well characterized. Of these PARP3 has been shown to have a distinct role in DNA repair. This protein has been shown to have a critical role in DSB resolution and to interact with partner proteins know to function in classical nonhomologous end joining (NHEJ) such as DNA-PKcs, Ku70, and Ku80. In order to examine the role of PARP3 vis-à-vis chemotherapy, we utilized the specific PARP3 inhibitor, ME-0328 in combination with activated cyclophosphamide (4HC) in the chronic lymphocytic leukaemia (CLL) cell lines, MEC1 and MEC2. Results from MTT cytotoxicity assays showed that the 50% inhibitory concentration (IC50) (50% of control) of 4HC in MEC-1 cells was 12.9μM. When used in combination with a nontoxic concentration (2μM) of the specific PARP3 inhibitor ME-0328 the IC50 of 4HC was reduced to 3.3μM. In MEC-2 cells the IC50 of 4HC was 10.8μM and was reduced to 2.1μM in the presence of ME-0328 at 2μM. ABT-888, a specific PARP1/2 inhibitor in clinical trials had a minimal effect on 4HC cytotoxicity in these cell lines. 4HC results in interstrand crosslinks (ICLs) which are believed to be repaired by homologous recombination. Intriguingly, PARP3 negatively regulates class switch recombination via activation-induced cytidine deaminase in B-lymphocytes. This may be involved in ME-0328 sensitization of 4HC in B-lymphocytic malignancies. Given these preliminary results we hypothesize that the PARP3 inhibitor, ME-0328 will sensitize B-lymphocytic cancers to chemotherapies. To explore this hypothesis this project will pursue the following objectives: 1. Characterize the selective sensitization of ME-0328 with 4HC and/or bendamustine (an ICL-inducing drug utilized in the treatment of B-lymphocyte malignancies) in B-lymphoma and CLL cell lines plus CLL clinical samples. 2. Determine the mechanisms by which ME-0328 sensitizes CLL cells to 4HC and/or bendamustine (i.e. reduction of PARP1 or 3 activity and/or altered activity of known DNA repair proteins (Rad51, DNA-PK, H2AX) and/or altered activation-induced cytidine deaminase). 3. Determine the in-vivo activity of 4HC and/or bendamustine with or without ME-0328 in a Rag2 /-γcc-/- xenograft model using the MEC1-CLL cell line. The inhibition of PARP3 may increase the sensitivity of tumor cells to DNA damaging chemotherapies. Our results should stimulate development of specific PARP3 inhibitors for clinical use. Citation Format: Bahram Sharif-Askari, David Davidson, Lilian Amrein, Lawrence Panasci. PARP3 inhibitors in cancer therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 268.

723 - Radio-sensitizing effects of all trans retinoic acid (ATRA) on human chronic lymphocytic leukemia and osteosarcoma cell lines

723 - Radio-sensitizing effects of all trans retinoic acid (ATRA) on human chronic lymphocytic leukemia and osteosarcoma cell lines

Study of DNA demethylation acting on DNA methyltransferase gene and microRNA in B-cell acute lymphocytic leukemia cell line

Objective To explore the function of 5-Aza-CdR in B-cell acute lymphocytic leukemia cell line NALM-6 and its influence on the expression of microRNA (miRNA) in the cells. Methods NALM-6 was treated with different concentrations of 5-Aza-CdR. Cell proliferation was detected by methyl thiazolyl tetrazolium (MTT) test, and DNA methyltransferase (DNMT) mRNA expression level was detected by reverse transcription PCR (RT-PCR). The expression changes of miRNA were detected by miScript miRNA PCR Array chip in cells after methylation. Results NALM-6 cell growth was inhibited by different concentrations of 5-Aza-CdR processing time, reaching to the maximum inhibitory rate was (74.163±0.381)%. 5-Aza-CdR affected concentrations was inversely proportional with expression level of DNMT mRNA. After 1 000 μmol/L of 5-Aza-CdR was dealed with cell 72 h, the relative expression of DNMT-1 was reduced to 0.453±0.021, DNMT-3L was 0.003±0.001, DNMT-3B was 0.395±0.019. MiScript miRNA PCR array sieved out 3 miRNA (miR-184, miR-23a-3p, miR-34a-5p) associated with DNA methylation. Conclusions 5-Aza-CdR down regulates the expression of DNMT gene in NALM-6 cells, and inhibits the proliferation of cells. MiR-184, miR-23a-3p and miR-34a-5p are related to DNA methylation in the occurrence and development of B-cell acute lymphocytic leukemia. Key words: Leukemia, B-cell lymphocytic, acute; DNA methylation; MicroRNA; 5-Aza-CdR

Effect of Emodin Derivative E11 on T Lymphocytic Leukemia Cell Line Molt-4 and Its Possible Mechanisms

To explore the effect of a new emodin derivative E11 on proliferation and apoptosis of T lymphocytic leukemia cell line Molt-4 and its possible mechanisms. MTT method was used to plot cell growth curve. Colony culture assay was performed for studying the effect of emodin derivative E11 on colony-formation of Molt-4. The fluorescent microscopy with DAPI staining was used to examine the cell morphological changes after E11 treatment. DNA fragmentation method was used to detect the inducing effect of emodin derivative E11 on cell apoptosis. Western blot was used to determine the expressions of apoptosis-related proteins including procaspase-9, procaspase-3, PARP and PI3K/AKT, MAPK signalling pathway. Emodin derivative E11 could strongly inhibit the growth of Molt-4 with the IC50 in 48 h at 1.381 ± 0.1552 µmol/L in dose-dependent manner. 0.1 µmol/L of E11 could inhibit cell colony formation. The typrical apopototic morphologic changes of Molt cells treated with E11 could be observed under fluorescence microscope with DAPI staining. DNA apoptotic ladder could be observed by DNA fragmentation.The expressions of procaspase -9, procaspase-3, PARP, p-MAPK, p-AKT, mTOR, p-mTOR, p-P70 and p-4BEP1 were down-regulated, while expressions of MAPK, AKT, 4EBP1 and P70 were not changed remarkably after Molt-4 were treated with E11 for 48 h. E11 can remarkably inhibit the proliferation and induce the apoptosis of Molt-4 cells. The mechanism of apoptosis of Molt-4 cells may be related with the suppression of PI3K/AKT and MAPK signalling pathways.

A microfluidic platform enabling single-cell RNA-seq of multigenerational lineages.

We introduce a microfluidic platform that enables off-chip single-cell RNA-seq after multi-generational lineage tracking under controlled culture conditions. We use this platform to generate whole-transcriptome profiles of primary, activated murine CD8+ T-cell and lymphocytic leukemia cell line lineages. Here we report that both cell types have greater intra- than inter-lineage transcriptional similarity. For CD8+ T-cells, genes with functional annotation relating to lymphocyte differentiation and function—including Granzyme B—are enriched among the genes that demonstrate greater intra-lineage expression level similarity. Analysis of gene expression covariance with matched measurements of time since division reveals cell type-specific transcriptional signatures that correspond with cell cycle progression. We believe that the ability to directly measure the effects of lineage and cell cycle-dependent transcriptional profiles of single cells will be broadly useful to fields where heterogeneous populations of cells display distinct clonal trajectories, including immunology, cancer, and developmental biology.

Potentiation of luteolin cytotoxicity by flavonols fisetin and quercetin in human chronic lymphocytic leukemia cell lines.

Despite numerous studies chronic lymphocytic leukemia (CLL) still remains an incurable disease. Therefore, all new compounds and novel strategies which are able to eradicate CLL cells should be considered as valuable clues for a potential future remedy against this malignancy. In the present study, the cytotoxic profiles of natural flavonoids were described in two human CLL cell lines, HG-3 and EHEB, indicating the flavone luteolin as the most potent flavonoid with half-maximal inhibitory constants (IC50) of 37 μM and 26 μM, respectively. Luteolin significantly increased the apoptotic cell population in both cell lines by increasing the activities of caspases-3 and -9 and triggering the intrinsic apoptotic pathway. Two flavonols, fisetin and quercetin, were somewhat less efficient in suppressing cellular viability, whereas baicalein, chrysin, (+)-catechin and hesperetin exerted only a small or no response at doses as high as 100 μM. Both fisetin and quercetin were able to augment the cytotoxic activity of luteolin in both cell lines by reducing the IC50 values up to four fold. As a result of this, luteolin displayed cytotoxicity activity already at low micromolar concentrations that could potentially be physiologically achievable through oral ingestion. No other tested flavonoids were capable of sensitizing CLL cells to luteolin pointing to a specific binding of fisetin and quercetin to the cellular targets which interfere with the signaling pathways induced by luteolin. Although further molecular studies to unravel this potentiating mechanism are certainly needed, this phenomenon could contribute to future remedies for prevention and treatment of chronic lymphocytic leukemia.

Transfection CXCR3 Gene into Lymphocytic Leukemia Cell Lines By Lentiviral Vector

Objective: Chemokine receptors and their ligands play important roles in the leukemia-initiating cell activity ,BCP-ALL progression and CNS infiltrating. To study the role of chemokine receptor CXCR3 in the mechanism of leukemia cell infiltration of the CNS, CXCR3 overexpression cell lines should be established. Lentivirus generally does not interfere with proto-oncogene and can express long-term and stable, so it is a good vector for genetic research on hematopoietic cells and T cells.Methods: Mouse CXCR3 gene fragment was extracted by PCR from T vector which was kept in our lab. After the extraction, CXCR3 fragment and linearized LV5 lentivirus overexpressing vector(with GFP+-puro) were digested with NotI and BamHI.After this,we used T4 DNA ligase to connect them, making the target gene (CXCR3) cloned into LV5 vector, to obtain recombinant shuttle plasmid. Next,we mixed the shuttle plasmid with packaging plasmids (pGag/Pol, pRev, pVSV-G), packaged lentivirus, collected, tested viral titer and sequenced target gene by DNA sequencing. Then the constructed CXCR3 overexpression lentivirus transfected Jurkat and L1210 cell lines, at the same time MOI achieved to 100, twice infection and adding polybrene 5ug/ml to increase lentivirus transfection efficiency. After GFP expression was observed under fluorescent microscope, we used puromycin to select transfected cells and tested the expression of CXCR3 on cell surface by flow cytometry analysis of Anti-Mouse CD183 (CXCR3).Results: After we sequenced the target mouse CXCR3 gene, viral titer reached to 1×10^9 TU/ml and GFP expression was observed after transfection 96h (<10%) on lymphocytic leukemia cell lines. Through puromycin selection, GFP+ cells surpassed 90% and CXCR3 overexpression group had upregulated 30%-90% compared with control group.Conclusion: Chemokine receptor CXCR3 had been successful transfected into lymphocytic leukemia cell lines and had stably high expression. The effect of CXCR3 in leukemic invasion and leukemia progression can be further studied using constructed CXCR3 overexpression lymphocytic cell lines. DisclosuresNo relevant conflicts of interest to declare.

The effect of hydroxybenzoate calcium compounds in inducing cell death in epithelial breast cancer cells

&lt;span&gt;Hydroxybenzoate (HB) compounds have shown their significance in inducing apoptosis in primary chronic lymphocytic leukemia (CLL) and cancer cell lines, including HT-1080. The current study focuses on assessing the effects of 2-, 3- and 4-hydroxybenzoate calcium (HBCa) compounds on MCF-10A, MDA-MB231 and MCF-7 epithelial breast cell lines. The HBCa-treated cells were examined using annexin V, to measure apoptosis in the three epithelial breast cell lines, after 48 h of treatment. The results indicated that 0.5 and 2.5 mmol/L of HBCa induced cell death in a dose-dependent manner. The induction of cell death in normal MCF-10A cells was found to be significantly less (&lt;em&gt;p&lt;/em&gt; = 0.0003&lt;/span&gt;&lt;span&gt;–&lt;/span&gt;&lt;span&gt;0.0068), in comparison to the malignant cell lines (MDA-MB231 and MCF-7). HBCa compounds were also found to cause cell cycle arrest in the epithelial breast cells at G1/G0. Furthermore, HBCa compounds induced the upregulation of apoptotic proteins (p53, p21, Bax and caspase-3), as well as the downregulation of the anti-apoptotic protein Bcl-2, which may suggest that apoptosis is induced via the intrinsic pathway.&lt;/span&gt;

Abstract LB-306: An ensemble approach to accurately detect somatic mutations via adaptive boosting

Abstract Identifying somatic mutations is a key analysis in cancer research. The challenge lies in the impure and heterogeneous nature of the tumor samples. Oftentimes, an algorithm works well for one tumor but poorly for another. Here, we present an ensemble approach that integrates multiple algorithms and demonstrate its performance and high accuracy with validation from both synthetic data and real data. Our approach incorporates state-of-the-art callers including MuTect, SomaticSniper, VarScan2, JointSNVMix2, and VarDict for somatic mutation detection. Each of these algorithms has its unique strength, capable of detecting variants that are missed by some others. The call sets are combined based on 70 independent sequencing and genomic features, which are then used by an adaptively boosted decision tree learner. The learner is trained with a sophisticated simulated data to discriminate true mutations from very noisy data of the tumor samples. In our latest submission to the ICGC-TCGA DREAM Mutation Calling Challenge (the Challenge), our approach obtained an unprecedented somatic SNV detection accuracy of 97.1% with a recall of 94.2% and a precision of 99.9%. The synthetic data was a tumor-normal pair of samples with 30x sequencing depth each. The tumor sample was synthesized by spiking in a whole spectrum of variants ranging from SNVs/Indels to SVs, resulting in an SNV allele frequency (VAF) of 25%. We further validated our approach with “in silico titration”. The titration mixed two different real genomes at different proportions with validated ground truths to generate different sample conditions, ranging from the simplest case where the normal and tumor were pure to the more challenging case where the tumor and normal tissues cross contaminated. From an VAF of 50%, 25% to 15%, our approach achieved an accuracy of 95.7%, 92.5%, and 85.3% respectively based on cross validation, consistent with the results from the Challenge. Finally, we validated our approach with three widely-used and published cancer datasets, obtained from TCGA and EGA, including a whole-genome sequenced malignant melanoma cell line, a whole-genome sequenced chronic lymphocytic leukemia cell line, and a whole-exome sequenced colon adenocarcinoma patient sample with experimentally validated somatic mutations. Our approach was trained on the data from the Challenge and applied to the aforementioned samples to measure its accuracy. Our results showed that we achieved a recall of 98.9%, 89.1% and 87.9% respectively. Although precision on real data cannot be measured without a comprehensive whole-genome experimental validation, our comparatively smaller call sets compared to all other methods considered implying that it has the highest precision among all. We extended our study of the above three validation approaches, namely synthetic genomes, in silico titration, and real samples, to compare with all the five individual callers for accuracy performance. We found that our approach had the highest accuracy when compared to any individual caller. To conclude, our approach is shown to have high accuracy in different types and conditions of tumor samples and by far the best in its class. Citation Format: Li Tai Fang, Pegah T. Afshar, John C. Mu, Narges Bani Asadi, Wing H. Wong, Hugo Y. K. Lam. An ensemble approach to accurately detect somatic mutations via adaptive boosting. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-306. doi:10.1158/1538-7445.AM2015-LB-306

AGS67E, an Anti-CD37 Monomethyl Auristatin E Antibody-Drug Conjugate as a Potential Therapeutic for B/T-Cell Malignancies and AML: A New Role for CD37 in AML.

CD37 is a tetraspanin expressed on malignant B cells. Recently, CD37 has gained interest as a therapeutic target. We developed AGS67E, an antibody-drug conjugate that targets CD37 for the potential treatment of B/T-cell malignancies. It is a fully human monoclonal IgG2 antibody (AGS67C) conjugated, via a protease-cleavable linker, to the microtubule-disrupting agent monomethyl auristatin E (MMAE). AGS67E induces potent cytotoxicity, apoptosis, and cell-cycle alterations in many non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia (CLL) cell lines and patient-derived samples in vitro. It also shows potent antitumor activity in NHL and CLL xenografts, including Rituxan-refractory models. During profiling studies to confirm the reported expression of CD37 in normal tissues and B-cell malignancies, we made the novel discovery that the CD37 protein was expressed in T-cell lymphomas and in AML. AGS67E bound to >80% of NHL and T-cell lymphomas, 100% of CLL and 100% of AML patient-derived samples, including CD34(+)CD38(-) leukemic stem cells. It also induced cytotoxicity, apoptosis, and cell-cycle alterations in AML cell lines and antitumor efficacy in orthotopic AML xenografts. Taken together, this study shows not only that AGS67E may serve as a potential therapeutic for B/T-cell malignancies, but it also demonstrates, for the first time, that CD37 is well expressed and a potential drug target in AML.

Establishment and Characterization of PCL12, a Novel CD5+ Chronic Lymphocytic Leukaemia Cell Line.

Immortalized cell lines representative of chronic lymphocytic leukemia (CLL) can assist in understanding disease pathogenesis and testing new therapeutic agents. At present, very few representative cell lines are available. We here describe the characterization of a new cell line (PCL12) that grew spontaneously from the peripheral blood (PB) of a CLL patient with progressive disease and EBV infection. The CLL cell origin of PCL12 was confirmed after the alignment of its IGH sequence against the “original” clonotypic sequence. The IGH gene rearrangement was truly unmutated and no CLL-related cytogenetic or genetic lesions were detected. PCL12 cells express CD19, CD20, CD5, CD23, low levels of IgM and IgD and the poor-outcome-associated prognostic markers CD38, ZAP70 and TCL1. In accordance with its aggressive phenotype the cell line is inactive in terms of LYN and HS1 phosphorylation. BcR signalling pathway is constitutively active and anergic in terms of p-ERK and Calcium flux response to α-IgM stimulation. PCL12 cells strongly migrate in vitro in response to SDF-1 and form clusters. Finally, they grow rapidly and localize in all lymphoid organs when xenotrasplanted in Rag2-/-γc-/- mice. PCL12 represents a suitable preclinical model for testing pharmacological agents.