Human T-acute Lymphoblastic Leukemia Cells Research Articles

More Related Gene Pathways to Vincristine-Induced Death Events in a Human T-Acute Lymphoblastic Leukemia Cell Line.

Acute lymphoblastic leukemia (ALL) is common in children but rare in adults. Vincristine (VCR) is one of the drugs used at the beginning of treatment. Some genes are resistant to VCR in B-ALL. Here, we examined the effect of VCR on gene expression changes in a T-ALL cell line, Jurkat. The MTT method was used to determine the IC50 in Jurkat cells treated with different concentrations of VCR for 48 and 72 hours. Total RNA was isolated from the cells and cDNA was prepared. The Human Cancer Drug Target PCR Array kit was used to evaluate the 84 gene expression changes in Jurkat cells. Protein-protein interaction was analyzed by STRING software. We identified 66 differentially expressed genes as comparison to untreated cells. The response to VCR-induced apoptotic events was remarkable in the pathways of heat shock protein, topoisomerases, protein kinases, cathepsins and cell cycle. In other pathways, there were resistant genes as well as sensitive genes to VCR treatment. Some proteins like HSP90AA1 and ESR1 had determining associations with other proteins. The results suggest VCR target genes in T-ALL cells may be beneficial biomarkers for ALL treatment and can be used to select appropriate synergistic drugs for VCR.

Abstract 3613: Opposite effects of adipocytes and adenosine on hematological cancer cell survival and proliferation in vitro

Abstract Background: Tumor microenvironment is composed of stroma that contains adipocytes. It is well established that obesity is a cancer risk factor and is associated with a poor prognosis in cancer patients. Cancer-associated adipocytes are thought to interact with cancer cells to promote tumor progression. Moreover, adipocytes express CD73 and can produce adenosine which also has been shown to promote cancer development through its interaction with adenosine receptors on solid cancer cells. As hematological malignancies can proliferate in bone marrow which is rich in adipocytes, we hypothesized that these latter could influence cancer cell survival and proliferation through the action of adenosine. Methods: CD73 enzymatic activity was determined on human pre-adipocyte cells (hMAD, ASC) by the Malachite Green Assay. The expression of adenosine receptor was assessed on human T-acute lymphoblastic leukemia cells (Jurkat) and follicular lymphoma cells (RL) by FACS analysis. Cancer cell proliferation and percentage of cell death were evaluated by FACS using CFSE staining and DAPI respectively. Survival and proliferation was evaluated in the absence or presence of human pre-adipocytes (hMAD, ASC) or adenosine with or without an adenosine deaminase inhibitor (deoxycoformycin 10 µM). Results: CD73 enzymatic activity was observed both in hMAD and ASC cells, while Jurkat and RL cells express the four adenosine receptors A1, A2A, A2B and A3. Hematological cancer cells tend to proliferate more when co-cultivated with pre-adipocytes (Jurkat 63,8% alone vs 69,2% with hMAD; RL 67,7% alone vs 73,9% with hMAD; Jurkat 62,4% alone vs 69,6% with ASC; RL 66,1% alone vs 74,8% with ASC). These cells also tend to have a lower rate of spontaneous cell death in same conditions (Jurkat 31,4% alone vs 23,6% with hMAD; RL 37,1% alone vs 30,1% with hMAD; Jurkat 32,7% alone vs 30,9% with ASC; RL 53,5% alone vs 36,0% with ASC). In contrast, we found that hematological cancer cells proliferate less in the presence of adenosine in a dose-dependent manner starting from 200 µM in media containing 1% serum (Jurkat 71,0% alone vs 68,6% with 200 µM adenosine and 47,6% with 200 µM adenosine + deoxycoformycin; RL 79,0% alone vs 73,3% with 200 µM adenosine and 59,8% with 200 µM adenosine + deoxycoformycin). Finally, adenosine also induced more cell death in these cells (Jurkat 28,6% alone vs 41,2% with 200 µM adenosine and 57,4% with 200 µM adenosine + deoxycoformycin; RL: 28,2% alone vs 47,9% with 200 µM adenosine + deoxycoformycin). Conclusion: Adipose cells have pro-proliferative and protective effects on hematological cancer cells in vitro while acute exposure to adenosine at supraphysiological concentrations has a cytotoxic effect. These results do not suggest that adipocytes favor proliferation through adenosine signaling. Citation Format: Cécile Bossard, Charles Dumontet, Lars P. Jordheim. Opposite effects of adipocytes and adenosine on hematological cancer cell survival and proliferation in vitro. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3613. doi:10.1158/1538-7445.AM2014-3613

A Genetic Screen In Zebrafish Identified Dlst As a Potential Therapeutic Target For Human Acute T-Lymphoblastic Leukemia

Despite improvements in treatment for T-Acute Lymphoblastic Leukemia (T-ALL), only 70-80% of children and 40% of adults achieve long-term remission. To discover novel targets for the development of molecular therapeutics, we conducted a dominant genetic modifier screen to identify genes whose inactivation delays the onset of leukemia. We have generated a Myc-driven zebrafish model of T-ALL that recapitulates its human counterpart genetically and pathologically. Utilizing this zebrafish model of T-ALL, we conducted a genetic screen that identified a gene encoding dihydrolipoamide S-succinyltransferase (DLST), whose heterozygous loss significantly delays the onset of leukemia in zebrafish. DLST is the E2 component of α-ketogluterate dehydrogenase complex (KGDHC), a key enzyme in the TCA cycle that converts α-ketogluterate (α-KG) to Succinyl CoA. Western blot analysis revealed that DLST was upregulated in zebrafish tumor cells, T-ALL cell lines and primary human T-ALL samples. Zebrafish T-ALL cells with heterozygous inactivation of dlst have decreased cell size and slowed cell cycle progression, compared to T-ALL cells without dlst inactivation. To demonstrate that DLST is a relevant target in human T-ALL, we used two structural analogs of α-KG (α-keto-n-valeric acid sodium salt (KV) and α-keto-β-methyl-n-valeric acid (KMV)) to inhibit the KGDHC activity. Inhibition of KGDHC by both KV and KMV decreased cell viability and promoted apoptotic cell death in a panel of human T-ALL cell lines. Genetic inactivation of DLST, via shRNA knockdown, also decreased cell viability in MOLT16 human T-ALL cell line, confirming the therapeutic potential of DLST in human T-ALL treatment. In vivo treatment of our zebrafish T-ALL model with KV promoted tumor regression within seven days of treatment. Taken together our findings indicate that DLST is an important contributor to T-ALL pathologenesis, and suggest that DLST is a potential target for further therapeutic development to treat human T-ALL. Disclosures:No relevant conflicts of interest to declare.

Chemokine receptors as novel targets of the oncogene Notch1 in acute lymphoblastic leukemia.

7060 Background: Malignant cells from different cancers express different profiles of chemokine receptors (CKR). Their presence may influence site-specific spread of tumor cells, by enabling them to respond to chemokine gradient, and may increase cell sensibility to chemokine mediated proliferative and anti-apoptotic stimuli. Notch ability to positively regulate CKR has been reported: stimulation of Pax5-/- pre-B cells with the Notch ligand Delta-1 results in induction of transcripts for CCR4, CCR8 and CXCR 6; the Delta-1-dependent regulation of Langerhans cell development includes induction of CCR6 expression resulting in the activation of chemotactic response to MIP-1a; Notch controls CCR7 signaling a regulator of CNS infiltration in T-acute lymphoblastic leukemia (T-ALL). Methods: This work aims to explore the correlation between the activation of the Notch oncogenic pathway in T-ALL and multiple myeloma (MM) cells and the aberrant expression CKR. Human T-ALL cell lines were treated with the Notch activation inhibitor, DAPT, or with a potent inhibitor of the Notch target, C-MYC, and evaluated the expression and functions of CCR9, CCR5, and CXCR4. Results: Treatment of human T-ALL and MM cell lines with pharmacologic inhibitors of Notch receptor activation produced a significant reduction of CCR9, CCR5 and CXCR4 expression, at both mRNA and protein levels. Results were confirmed by chemotaxis and survival assays. We identified the product of C-MYC gene as a possible mediator of Notch effect in regulating CKR networks in T-ALL and MM. Conclusions: These results suggest that Notch receptors play a previously unknown role in cancer progression and metastasis, by maintaining the expression levels of CKR. In conclusion, the identification of the potential axis Notch/CKR could have a prognostic value and provide the rationale for a tailored approach, since both Notch and CKR are targeted by emerging drugs.

ERG promotes T-acute lymphoblastic leukemia and is transcriptionally regulated in leukemic cells by a stem cell enhancer

AbstractThe Ets-related gene (ERG) is an Ets-transcription factor required for normal blood stem cell development. ERG expression is down-regulated during early T-lymphopoiesis but maintained in T-acute lymphoblastic leukemia (T-ALL), where it is recognized as an independent risk factor for adverse outcome. However, it is unclear whether ERG is directly involved in the pathogenesis of T-ALL and how its expression is regulated. Here we demonstrate that transgenic expression of ERG causes T-ALL in mice and that its knockdown reduces the proliferation of human MOLT4 T-ALL cells. We further demonstrate that ERG expression in primary human T-ALL cells is mediated by the binding of other T-cell oncogenes SCL/TAL1, LMO2, and LYL1 in concert with ERG, FLI1, and GATA3 to the ERG +85 enhancer. This enhancer is not active in normal T cells but in transgenic mice targets expression to fetal liver c-kit+ cells, adult bone marrow stem/progenitors and early CD4−CD8− double-negative thymic progenitors. Taken together, these data illustrate that ERG promotes T-ALL and that failure to extinguish activity of stem cell enhancers associated with regulatory transcription factors such as ERG can contribute to the development of leukemia.

Notch3-mediated regulation of MKP-1 levels promotes survival of T acute lymphoblastic leukemia cells

Activation of the Notch pathway occurs commonly in T acute lymphoblastic leukemia (T-ALL) because of mutations in Notch1 or Fbw7 and is involved in the regulation of cell proliferation and survival. Deregulated Notch3 signalling has also been shown to promote leukemogenesis in transgenic mice, but the targets of Notch3 in human T-ALL cells remain poorly characterized. Here, we show that Notch3 controls levels of mitogen-activated protein kinase (MAPK) phosphatase 1 (MKP-1). In a model of T-ALL cell dormancy, both Notch3 activation and MKP-1 expression were upregulated in aggressive compared with dormant tumors, and this inversely correlated with the levels of phosphorylated p38 and extracellular signal-regulated kinase1/2 (ERK1/2) MAPKs, two canonical MKP-1 targets. We demonstrate that MKP-1 protein levels are regulated by Notch3 in T-ALL cell lines because its silencing by RNA interference or treatment with γ-secretase inhibitors induced strong MKP-1 reduction whereas activation of Notch3 signalling had the opposite effect. Furthermore, MKP-1 has an important role in T-ALL cell survival because its attenuation by short hairpin RNA significantly increased cell death under stress conditions. This protective function has a key role in vivo, as MKP-1-deficient cells showed impaired tumorigenicity. These results elucidate a novel mechanism downstream of Notch3 that controls the survival of T-ALL cells.

CD90 and CD110 correlate with cancer stem cell potentials in human T-acute lymphoblastic leukemia cells

Although cancer stem cells (CSCs) have been recently identified in myeloid leukemia, published data on lymphoid malignancy have been sparse. T-acute lymphoblastic leukemia (T-ALL) is characterized by the abnormal proliferation of T-cell precursors and is generally aggressive. As CD34 is the only positive-selection marker for CSCs in T-ALL, we performed extensive analysis of CD markers in T-ALL cell lines. We found that some of the tested lines consisted of heterogeneous populations of cells with various levels of surface marker expression. In particular, a small subpopulation of CD90 (Thy-1) and CD110 (c-Mpl) were shown to correlate with stem cell properties both in vitro and in transplantation experiments. As these markers are expressed on hematopoietic stem cells, our results suggest that stem cell-like population are enriched in CD90+/CD110+ fraction and they are useful positive-selection markers for the isolation of CSCs in some cases of T-ALL.

Suppressive activity of protease inhibitors from buckwheat seeds against human T-acute lymphoblastic leukemia cell lines.

The buckwheat protease inhibitor designated BWI-1, a member of the potato inhibitor I family, inhibits trypsin, chymotrypsin, and subtilisin, whereas the buckwheat protease inhibitor designated BWI-2a, a novel protease inhibitor homologous to the vicilin family, inhibits only trypsin. We examined the suppressive activity of BWI-1 and BWI-2a against T-acute lymphoblastic leukemia (T-ALL) cells, such as JURKAT and CCRF-CEM, and human normal blood lymphocytes. Both inhibitors significantly suppressed the growth of T-ALL cells as judged by the soluble 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (tetrazolium/formazan assay). JURKAT cells showed slightly higher susceptibility to buckwheat inhibitors than CCRF-CEM cells. Modification of Arg residue(s) in inhibitors by 1,2-cyclohexandione inactivated their trypsin inhibitory activity, considerably abolishing their suppressive activity. This suggests that the trypsin inhibitory activity is involved in the suppression of growth of human T-ALL cell lines. It was further found that both inhibitors triggered programmed cell death (apoptosis) of these cell strains with DNA fragmentation.

Establishment of a human T-acute lymphoblastic leukemia cell line with a (16;20) chromosome translocation

A new T-cell line. Loucy, was established from the peripheral blood of a patient with T-cell acute lymphoblastic leukemia (T-ALL). The surface marker analysis of the cell line is OKT3+, OKT4+, THB4+, J5±, OKT6−, TdT−, and HLA-DR−. indicating stage IV in T-cell lineage. Karyotype analysis revealed 45,X.5q−,t(16;20)(p12;q13). The translocation between chromosomes 16 and 20 has not been previously detected in ALL. This cell line may be of value in evaluating the role of t(16;20) in the etiology of T-ALL.