Human T-cell Acute Leukemia Research Articles

Validity of the cell-extracted proteome as a substrate pool for exploring phosphorylation motifs of kinases.

Three representative protein kinases with different substrate preferences, ERK1 (Pro-directed), CK2 (acidophilic), and PKA (basophilic), were used to investigate phosphorylation sequence motifs in substrate pools consisting of the proteomes from three different cell lines, MCF7 (human mammary carcinoma), HeLa (human cervical carcinoma), and Jurkat (human acute T-cell leukemia). Specifically, recombinant kinases were added to the cell-extracted proteomes to phosphorylate the substrates in vitro. After trypsin digestion, the phosphopeptides were enriched and subjected to nanoLC/MS/MS analysis to identify their phosphorylation sites on a large scale. By analyzing the obtained phosphorylation sites and their surrounding sequences, phosphorylation motifs were extracted for each kinase-substrate proteome pair. We found that each kinase exhibited the same set of phosphorylation motifs, independently of the substrate pool proteome. Furthermore, the identified motifs were also consistent with those found using a completely randomized peptide library. These results indicate that cell-extracted proteomes can provide kinase phosphorylation motifs with sufficient accuracy, even though their sequences are not completely random, supporting the robustness of phosphorylation motif identification based on phosphoproteome analysis of cell extracts as a substrate pool for a kinase of interest.

Abstract NG14: Dissecting immune microenvironment of T-cell acute lymphoblastic leukemia

Abstract NG14: Dissecting immune microenvironment of T-cell acute lymphoblastic leukemia

ЦИТОТОКСИЧНОСТЬ ПИРАНОПИРИДОНОВ С ТРИПТАМИНОВЫМ ФРАГМЕНТОМ

New derivatives of pyrano[3,2-c]pyridones III 5, 10, 15, 18 were synthesised by threecomponent reaction of pyridonotryptamines I, aromatic aldehydes II and malononitril. Pyridonotryptamines I were synthesised by reaction of tryptamines (these compounds were obtained by Grandberg reaction of arylhydrazines with γ-halogencarbonyl compounds) with 4-hydroxy- 6-methyl-2H-pyran- 2-on. The aromatic aldehydes II 1, 5, 6, 9 are commercially available compounds. Boiling a mixture of compounds I, II and malononitril (molar ratio 1:1.1:1.1) in ethyl alcohol in the presence of triethylamine gives the target compounds III 5, 10, 15, 18. The product yields 44–75%. The structure of pyrano[3,2-c]pyridones with tryptamine fragment III (four compounds) was proved by 1H NMR and confirmed by elemental analysis. Compounds III 1–4, 6–9, 11–14, 16 and 17 were synthesised earlier by a similar procedure. The cytotoxicity of the synthesised compounds III 1–18 was determined in vitro using the MTT test on human cell cultures A549 (lung carcinoma) and HCT116 (colorectal carcinoma). Camptothecin and daunorubicin were used as reference drugs. The value of the concentration inducing 50% inhibition of the cell growth (IC50, μM) was determined from the dose-response curves using GraphPad Prism 9 software. Compounds III 3 (R1=R2=Br, Ar=2,5-di-OMe-С6Н3,) III 4 (R1=R2=Br, Ar=4-F-С6Н4), III 6 (R1=Ме, R2=Н, Ar=2,4,5-tris-OMe-С6Н2), III 13 (R1=R2=Br, Ar=Ph), III 14 (R1=R2=Br, Ar=2,3-di-OMe-С6Н3) and III 16 (R1=R2=Br, Ar=Py) showed the best results for A549 and HCT116 cultures. The effect of the most active compounds III 3 and III 4 on the cell cycle and apoptosis was studied on Jurkat cell culture (human acute T-cell leukemia). Compounds III 3 and III 4 showed significant cytotoxicity against the Jurkat line in the resazurin test – 1.47 ± 0.06 and 4.56 ± 0.19 μM, respectively, comparable to the cytotoxicity of the reference drug camptothecin of 1.24 ± 0.05 μM. Based on the flow cytometry results, it is suggested that the effect is manifested by some (possibly reversible) cell cycle arrest in the presynthetic phase.

HSF1-Mediated Control of Cellular Energy Metabolism and mTORC1 Activation Drive Acute T-Cell Lymphoblastic Leukemia Progression.

Deregulated oncogenic signaling linked to PI3K/AKT and mTORC1 pathway activation is a hallmark of human T-cell acute leukemia (T-ALL) pathogenesis and contributes to leukemic cell resistance and adverse prognosis. Notably, although the multiagent chemotherapy of leukemia leads to a high rate of complete remission, options for salvage therapy for relapsed/refractory disease are limited due to the serious side effects of augmenting cytotoxic chemotherapy. We report that ablation of HSF1, a key transcriptional regulator of the chaperone response and cellular bioenergetics, from mouse T-ALL tumors driven by PTEN loss or human T-ALL cell lines, has significant therapeutic effects in reducing tumor burden and sensitizing malignant cell death. From a mechanistic perspective, the enhanced sensitivity of T-ALLs to HSF1 depletion resides in the reduced MAPK-ERK signaling and metabolic and ATP-producing capacity of malignant cells lacking HSF1 activity. Impaired mitochondrial ATP production and decreased intracellular amino acid content in HSF1-deficient T-ALL cells trigger an energy-saving adaptive response featured by attenuation of the mTORC1 activity, which is coregulated by ATP, and its downstream target proteins (p70S6K and 4E-BP). This leads to protein translation attenuation that diminishes oncogenic signals and malignant cell growth. Collectively, these metabolic alterations in the absence of HSF1 activity reveal cancer cell liabilities and have a profound negative impact on T-ALL progression. IMPLICATIONS: Targeting HSF1 and HSF1-dependent cancer-specific anabolic and protein homeostasis programs has a significant therapeutic potential for T-ALL and may prevent progression of relapsed/refractory disease.

Cytotoxic and Antioxidant Effects of Antimalarial Herbal Mixtures.

Many developing countries depend on herbal mixtures as the first line and cost-effective therapy for malaria. These mixtures with such curative tendencies may also be a source of toxicity to host cells. On the other hand, these mixtures may have anticancer potential activity characterized by cytotoxicity to cancer cells. The aim of the study was to determine the cytotoxic and antioxidant effects of five different antimalarial herbal mixtures. Five antimalarial herbal mixtures commonly used in Ghana (coded as STF, SMH, SMM, SGM, and STT) were purchased and freeze-dried. The dried samples were tested on human acute T-cell leukemia (Jurkat) and breast adenocarcinoma (MCF-7) cell lines. Cytotoxicity was assessed using the tetrazolium-based colorimetric (MTT) assay while antioxidant activity was determined using DPPH free-radical scavenging assay. Among the mixtures, SMM and SGM exhibited the strongest cytotoxicity towards Jurkat cells (IC50 values 59.17 μg/ml and 49.57 μg/ml, respectively), whereas STT showed the weakest cytotoxicity (IC50 = 244.94 μg/ml). Cytotoxic effect of SMM was also strongest towards MCF-7 cells whilst the least cytotoxic sample was SGM (IC50 > 1000 μg/ml). SMM had the highest antioxidant percentage (EC50 = 1.05 mg/ml). The increasing order of antioxidant percentage among the five herbal mixtures is SMM > SMH > STT > STF > SGM. The herbal mixtures may be potential sources of toxic agents to host cells. Therefore, further toxicity studies must be performed to safeguard health of the public. Interestingly, cytotoxicities exhibited by SMM and SGM suggest the presence of anticancer constituents in them which warrant further studies.

Caffeic acid and chlorogenic acid cytotoxicity, genotoxicity and impact on global DNA methylation in human leukemic cell lines.

Dietary phenolic compounds such as caffeic and chlorogenic acid exert an antiproliferative effect and modulate the gene-specific DNA methylation status in human breast tumor cells, but it remains unclear whether they interfere with global DNA methylation in human leukemia cells. We examined whether caffeic and chlorogenic acid (1-250 µM) exert antitumor action in human promyelocytic leukemia cells (HL-60) and human acute T-cell leukemia cells (Jurkat). Caffeic and chlorogenic acid did not reduce cell viability in the two cell lines, as assessed using the neutral red uptake and MTT assays. These phenolic acids (1-100 μM) neither induced DNA damage (comet assay) nor increased the micronuclei frequency (micronucleus assay) in HL-60 and Jurkat cells, indicating that they were not genotoxic or mutagenic. Analysis of global DNA methylation levels using a 5-mC DNA ELISA kit revealed that chlorogenic acid at a non-cytotoxic concentration (100 μM) induced global DNA hypomethylation in Jurkat cells, but not in HL-60 cells, suggesting that it exerts a cell-specific effect. Caffeic acid did not change global DNA methylation. As other phenolic compounds, chlorogenic acid probably modulates DNA methylation by targeting DNA methyltransferases. The hypomethylating action of chlorogenic acid can be beneficial against hematological malignances whose pathogenic processes involve impairment of DNA methylation.

Fli-1 promotes proliferation and upregulates NANOGP8 expression in T-lymphocyte leukemia cells

Friend leukemia integration 1 (Fli-1) is a member of the E26 transformation-specific (ETS) transcription factor family. Fli-1 regulates normal hematopoiesis and vasculogenesis, and its aberrant expression underlies virus-induced leukemias and various types of human cancers. NANOGP8, a retro-pseudogene of stem cell mediator NANOG, is expressed predominantly in cancer cells and plays a role in tumorigenesis. In this study, we demonstrate that Fli-1 expression enhances human acute T-cell leukemia Jurkat cell proliferation and that Fli-1 acts as a transcriptional activator of NANOGP8 expression in these cells. NANOGP8 and Fli-1 are highly expressed in Jurkat cells, whereas NANOG was undetectable at both the RNA and protein levels. Moreover, the expression of endogenous NANOGP8 was significantly influenced by gain of function and loss of function of Fli-1. Promoter-reporter assays showed that NANOGP8 transcription was significantly upregulated by dose-dependent Fli-1 overexpression. A series of deletion mutagenesis of NANOGP8 promoter sequence revealed that NANOGP8 promoter activity was tightly regulated and found the minimal promoter region sufficient to activate NANOGP8 transcription mediated by Fli-1. Moreover, site-directed mutagenesis of the putative binding site abolished both NANOGP8 full-length and minimal promoter activities. Binding assays revealed that Fli-1 directly interacts with the potent binding site in NANOG promoter region. Taken together, our data demonstrate that Fli-1 is a novel upstream transcriptional activator of NANOGP8 and provide the molecular details of Fli-1-mediated NANOGP8 gene expression. Ultimately, these findings may contribute to understanding the expanded regulatory mechanisms of oncogenic NANOGP8 and ETS family transcription factors in leukemogenesis.

Preparation of immobilized trypsin micro monolithic column and its application to rapid digestion of trace proteins

A method for the preparation of a micro monolithic column with immobilized trypsin was developed for the rapid and efficient digestion of proteins. The micro monolithic column was prepared by photo-polymerization inside the tip of 20 μL pipette. The polymerization solution was composed of the functional monomers 4-pentenoic succinimide ester (PAS) and 2-hydroxyethyl methacrylate (HEMA), crosslinker pentaerythritol triacrylate (PETA) dissolved in a ternary porogenic system comprising dimethyl sulfoxide (DMSO), formyldimethylamine (DMF) and 1-dodecanol. Immobilization of trypsin was achieved by a chemical reaction between the amino group and succinimide. The effects of the active ester content in the polymerization mixture and the volumes of the monolithic bed on the column capacities of the immobilized trypsin were systematically investigated. The digestion efficiency as well as the stability and repeatability of the immobilized trypsin were systematically investigated by using standard proteins cytochrome C and bovine serum albumin. The experimental results indicated that high digestion efficiency with great reproducibility between batches and the digestion procedure could be obtained within 10 min. The trypsin immobilized columns were applied to the digestion of proteins extracted from 1×105 human acute promyelocytic leukemia (NB4) cells and human acute T-cell leukemia (Jurkat T) cells. A total of 2489 and 2572 proteins were readily identified by Nano-LC-MS/MS analysis. The quantity ratio of the identified proteins increased 2.2% and 6.1%, respectively, compared to the case of in-solution digestion, demonstrating the robustness of the trypsin immobilized micro column and its potential application to proteome studies.

PTPN22 Silencing in Human Acute T-Cell Leukemia Cell Line (Jurkat Cell) and its Effect on the Expression of miR-181a and miR-181b.

Purpose: T-cell acute lymphoblastic leukemia (T-ALL) is one of the most common malignancies associated with T-lymphocytes, accounting for 10 to 15 percent of ALL cases in children and 25 percent in adults. Innovative therapeutic approaches that overcome ineffective treatments on tumor cells may be a potential source of improvement in therapeutic approaches. Suppression of gene expression at transfusion level is one of the important strategies in gene therapy. The expression of PTPN22 and miR-181 genes in all types of hematologic malignancies increases and is likely to contribute to the survival and death of cells by affecting a variety of signaling pathways. The purpose of this study was to determine the role of PTPN22 inhibition by siRNA, and alteration in miR-181a and miR-181b in Jurkat cell line.Methods: Jurkat cells were transfected with 80 pmol of siRNA to inhibit PTPN22. After that, expression of PTPN22 mRNA and transcript levels of miR-181a and miR-181b were measured with Real-time PCR after 48hrs.Results: Experiments demonstrated that siRNA transfection resulted in significant downregulation of PTPN22 mRNA after 48 hrs in 80 pmol dose of siRNA. Moreover, transcript levels of both miR-181a and miR-181b was decreased after transfection.Conclusion: PTPN22, miR-181a and miR-181b might be involved in progression of Jurkat cells and targeting these molecules by RNAi might confer promising tool in treatment of T-ALL.

Rocaglamide breaks TRAIL-resistance in human multiple myeloma and acute T-cell leukemia in vivo in a mouse xenogtraft model

Multiple myeloma (MM) is an incurable malignancy by the presently known therapies. TRAIL is a promising anticancer agent that virtually not shows any toxicity to normal cells. We have recently carried out clinical trials with a human circularly permuted TRAIL, CPT, against MM saw a partial response in approximate 20–30% of patients. In the current study, we investigated the cause of CPT resistance and revealed that the majority of the MM patients express elevated levels of c-FLIP. Knockdown of c-FLIP expression by siRNA alone was sufficient to increase CPT-mediated apoptosis in a CPT-resistant human MM cell line U266. To overcome CPT resistance, we investigated the combination of CPT with Rocaglamides(s) in MM which has been shown to inhibit c-FLIP expression in vitro. We show that Rocaglamide(s) overcomes CPT resistance in U266 in vitro and significant increases in anti-tumor efficacies of CPT in mice xenografted with U266. Similar results were also obtained in mice xenografted with the CPT-resistant human acute T-cell leukemia cell line Molt-4. Our study suggests that the combination of Rocaglamide(s) with CPT may provide a more efficient treatment against myeloma and leukemia.

Antiproliferative and proapoptotic effects of a pyrrole containing arylthioindole in human Jurkat leukemia cell line and multidrug-resistant Jurkat/A4 cells

Recently, a series of novel arylthioindole compounds, potent inhibitors of tubulin polymerization and cancer cell growth, were synthesized. In the present study the effects of 2-(1H-pyrrol-3-yl)-3-((3,4,5-trimethoxyphenyl)thio)-1H-indole (ATI5 compound) on cell proliferation, cell cycle progression, and induction of apoptosis in human T-cell acute leukemia Jurkat cells and their multidrug resistant Jurkat/A4 subline were investigated. Treatment of the Jurkat cells with the ATI5 compound for 48 hrs resulted in a strong G2/M cell cycle arrest and p53-independent apoptotic cell death accompanied by the induction of the active form of caspase-3 and poly(ADP-ribose) polymerase-1 (PARP-1) cleavage. ATI5 treatment also caused non-cell death related mitotic arrest in multidrug resistant Jurkat/A4 cells after 48 hrs of treatment suggesting promising opportunities for the further design of pyrrole-containing ATI compounds as anticancer agents. Cell death resistance of Jurkat/A4 cells to ATI5 compound was associated with alterations in the expression of pro-survival and anti-apoptotic protein-coding and microRNA genes. More importantly, findings showing that ATI5 treatment induced p53-independent apoptosis are of great importance from a therapeutic point of view since p53 mutations are common genetic alterations in human neoplasms.

Biosynthesis and Characterization of Silver Nanoparticles using Catharanthus roseus Leaf Extract and its Proliferative Effects on Cancer Cell Lines

Green synthesis is one of the most rapid, reliable, and best routes for the synthesis of silver nanoparticles (AgNPs). Previous studies demonstrated that active compounds in Catharanthus roseus were responsible for bioreduction during the synthesis of spherical AgNPs. In the present study, synthesis of AgNPs using C. roseus aqueous extract was optimized. To prepare the extract, dried C. roseus leaves were boiled at 100°C for 5 min or soaked in a water bath at 40°C for 24 h. The extracts at concentrations of 10% and 20% were mixed with AgNO3 solutions of 1 and 5 mM. The resulting AgNPs were characterized by ultraviolet-visible (UV-Vis), X-ray diffraction (XRD), and transmission electron microscope (TEM). Cell proliferation of Jurkat (human acute T-cell leukemia) and HT-29 (human colorectal adenocarcinoma) cell lines in solutions containing synthesized AgNPs or C. roseus aqueous extract was measured by the MTS [3-(4,5-dimethylthiazol-2- yl)-5-(3-carbonxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium/phenazine metho sulfate] assay using the double dilution approach (concentrations ranging from 3.91 to 1000 μg/ml). UV-Vis spectra analysis indicated that the AgNPs at 5 mM AgNO3 + 10% extract produced the highest surface Plasmon resonance peak at approximately 500 nm. The XRD analysis confirmed the presence of AgNPs with a face-centered cubic structure. TEM analysis showed that the AgNPs ranged in size from 20 to 50 nm with spherical shapes. Based on the MTS assay, the IC50 values of AgNPs ranged from 13.68 to 46.88 μg/ml for both cancer cell lines, whereas the IC50 values of C. roseus aqueous extract ranged from 62.50 to 312.50 μg/ml for the Jurkat cell line and no IC50 values for HT-29 cell lines. Further studies should be conducted to validate the potential application of AgNPs as anti-cancer agents.

Prognostic Significance of the Lymphoblastic Leukemia-Derived Sequence 1 (LYL1) GeneExpression in Egyptian Patients with AcuteMyeloid Leukemia.

Objective: Aberrant activation of transcription factor genes is the most frequent target of genetic alteration in lymphoid malignancies. The lymphoblastic leukemia-derived sequence 1 (LYL1) gene, which encodes a basic helix-loop helix, was first identified with human T-cell acute leukemia. Recent studies suggest its involvement in myeloid malignancies. We aimed to study the expression percent of oncogene LYL1 in primary and secondary high-risk myeloid leukemia and the impact on prognostic significance in those patients. Materials and Methods: Using quantitative real-time polymerase chain reaction for detection of LYL1 oncogenes, our study was carried out on 39 myeloid leukemia patients including de novo cases, myelodysplastic syndrome (MDS) with transformation, and chronic myelogenous leukemia (CML) in accelerated and blast crisis, in addition to 10 healthy individuals as the reference control. Results: LYL1 expression was increased at least 2 times compared to the controls. The highest expression of this transcription factor was observed in the MDS cases transformed to acute leukemia at 7.3±3.1, p=0.0011. LYL1 expression was found in 68.2%, 75%, and 77.8% of cases of acute myeloid leukemia, CML crisis, and MDS, respectively. Significant correlation of LYL1 overexpression with some subtypes of French-American-British classification was found. There was, for the first time, significant correlation between the blood count at diagnosis and LYL1 expression (p=0.023, 0.002, and 0.031 for white blood cells, hemoglobin, and platelets, respectively). The rate of complete remission was lower with very high levels of LYL1 expression and the risk of relapse increased with higher levels of LYL1 expression, suggesting an unfavorable prognosis for cases with enhanced expression.Conclusion: Overexpression of LYL1 is highly associated with acute myeloid leukemia and shows more expression in MDS with unfavorable prognosis in response to induction chemotherapy. These observations could signal a promising tool for a therapeutic target to basic helix–loop helix protein related to transcription factors, which may improve patient outcome in acute myeloid leukemia, MDS, and CML in blast crisis.

MIP-1α promotes the migration ability of Jurkat cell through human brain microvascular endothelial cell monolayer

This study was purposed to explore the mechanism of central nervous system (CNS) leukemia resulting from brain metastasis of human acute T-cell leukemia (T-ALL) cells and the role of MIP-1α in migration of Jurkat cells through human brain microvascular endothelial cells (HBMEC). The real-time PCR, siRNA test, transendothelial migration test, endothelial permeability assay and cell adhesion assay were used to detect MIP-1α expression, penetration and migration ability as well as adhesion capability respectively. The results showed that the MIP-1α expression in Jurkat cells was higher than that in normal T cells and CCRF-HSB2, CCRF-CEM , SUP-T1 cells. The MIP-1α secreted from Jurkat cells enhanced the ability of Jurkat cells to penetrate through HBMEC, the ability of Jurkat cells treated by MIP-1α siRNA to adhere to HBMEC and to migrate trans endothelial cells decreased. It is concluded that the MIP-1α secreted from Jurkat cells participates in process of penetrating the Jurkat cells through HBMEC monolayer.

Core Transcriptional Regulatory Circuit Controlled by the TAL1 Complex in Human T Cell Acute Lymphoblastic Leukemia

The oncogenic transcription factor TAL1/SCL is aberrantly expressed in over 40% of cases of human Tcell acute lymphoblastic leukemia (T-ALL), emphasizing its importance in the molecular pathogenesis of T-ALL. Here we identify the core transcriptional regulatory circuit controlled by TAL1 and its regulatory partners HEB, E2A, LMO1/2, GATA3, and RUNX1. We show that TAL1 forms a positive interconnected autoregulatory loop with GATA3 and RUNX1 and that the TAL1 complex directly activates the MYB oncogene, forming a positive feed-forward regulatory loop that reinforces and stabilizes the TAL1-regulated oncogenic program. One of the critical downstream targets in this circuitry is the TRIB2 gene, which is oppositely regulated by TAL1 and E2A/HEB and is essential for the survival of T-ALL cells.

Combined treatment with fenretinide and indomethacin induces AIF-mediated, non-classical cell death in human acute T-cell leukemia Jurkat cells

Currently used cytotoxic drugs in cancer therapy have a similar mechanism of action and low specificity. Applied simultaneously, they show an additive effect with strong side effects. Clinical trials with the use of different agents in cancer therapy show that the use of these compounds alone is not very effective in fighting cancer. An alternative solution could be to apply a combination of these agents, because their combination has a synergistic effect on some cancer cells. Therefore, in our investigations we examined the effects of a synthetic retinoid-fenretinide when combined with a non-steroidal anti-inflammatory drug–indomethacin on the process of apoptosis in the acute human T-cell leukemia cell line Jurkat. We demonstrate that treatment with the combination of the tested compounds induces the death of cells, that is peculiar and combines features of apoptosis as well as non-apoptotic cell death. In detail we observed, cell membrane permeabilization, phosphatydylserine exposure, no oligonucleosomal DNA fragmentation, no caspase-3 activation, but apoptosis inducing factor (AIF) nuclear translocation. Taken together these results indicate, that Jurkat cells after treatment with a combination of fenretinide and indomethacin undergo AIF-mediated programmed cell death.

Apoptosis in immunocytes induced by several types of pesticides

Several types of pesticides, such as organophosphates and organochlorines, can induce thymocyte apoptosis, resulting in thymic atrophy and predisposing the highly sensitive fetal immune system to loss of tolerance to self-antigens and subsequent increased risk for autoimmune disease and allergies. In the studies here, mouse primary thymocytes and a human acute T-cell leukemia cell line (J45.01) were employed to examine potential thymocyte apoptosis induced by several types of chemicals, including several commonly-used pesticides. Thymocytes and J45.01 cells were treated for 4 or 8 hr with varying doses of metamidophos, parathion, PNMC, or methoxychlor; dexamethasone was used as a positive control. Apoptosis, cell viability, the proportion of Annexin-V+ cells, the activities of caspases 3/7, 8, and 9, and the levels of DNA fragmentation in both the J45.01 cells and thymocytes were then examined. The results here show that with both cell types, there was an increase in the proportion of annexin-V+ cells and levels of DNA fragmentation following exposure to parathion, PNMC, methoxychlor, or dexamethasone (positive control); however, the levels of sensitivity appeared to differ between the cell types. Furthermore, caspase-7 and -8 activities also differed between the J45.01 cells and thymocytes when treated with PNMC, methoxychlor, or dexamethasone. A more precise characterization of these inter-cellular differences is the logical next step in our studies of the effects of these (and other) pesticides on immune cell integrity. These specific types of follow-on mechanistic experiments are currently underway in our laboratories.

Mutations in Multiple Domains of c-Myb Disrupt Interaction with CBP/p300 and Abrogate Myeloid Transforming Ability

The c-myb proto-oncogene is a key regulator of hematopoietic cell proliferation and differentiation. MYB mRNA is expressed at high levels in, and is required for the proliferation of, most human myeloid and acute lymphoid leukemias. Recently, chromosomal translocation and genomic duplications of c-MYB have been identified in human T-cell acute leukemia. The present work focuses on the effects of mutations in different domains of the murine c-Myb protein on its transforming ability as defined by suppression of myelomonocytic differentiation and continued proliferation. Using both a novel myeloid cell line-based assay and a primary hematopoietic cell assay, we have shown that mutation of single residues in the transactivation domain important for CBP/p300 binding leads to complete loss of transforming ability. We also simultaneously mutated residues in the DNA-binding domain and the negative regulatory domain of the protein. These double mutants, but not the corresponding single mutants, show a complete loss of transforming activity. Surprisingly, these double mutants show severely impaired transactivation and are also defective for CBP/p300 binding. Our results imply that multiple Myb domains influence its interaction with CBP/p300, highlight the importance of this interaction for myeloid transformation, and suggest an approach for molecular targeting of Myb in leukemia.

NOTCH is a key regulator of human T-cell acute leukemia initiating cell activity

AbstractUnderstanding the pathways that regulate the human T-cell acute lymphoblastic leukemia (T-ALL) initiating cells (T-LiC) activity has been hampered by the lack of biologic assays in which this human disease can be studied. Here we show that coculture of primary human T-ALL with a mouse stromal cell line expressing the NOTCH ligand delta-like-1 (DL1) reproducibly allowed maintenance of T-LiC and long-term growth of blast cells. Human T-ALL mutated or not on the NOTCH receptor required sustained activation of the NOTCH pathway via receptor/ligand interaction for growth and T-LiC activity. On the reverse, inhibition of the NOTCH pathway during primary cultures abolished in vitro cell growth and in vivo T-LiC activity. Altogether, these results demonstrate the major role of the NOTCH pathway activation in human T-ALL development and in the maintenance of leukemia-initiating cells.

The C-MYB locus is involved in chromosomal translocation and genomic duplications in human T-cell acute leukemia (T-ALL), the translocation defining a new T-ALL subtype in very young children

AbstractThe C-Myb transcription factor is essential for hematopoiesis, including in the T-cell lineage. The C-Myb locus is a common site of retroviral insertional mutagenesis, however no recurrent genomic involvement has been reported in human malignancies. Here, we identified 2 types of genomic alterations involving the C-MYB locus at 6q23 in human T-cell acute leukemia (T-ALL). First, we found a reciprocal translocation, t(6;7)(q23;q34), that juxtaposed the TCRB and C-MYB loci (n = 6 cases). Second, a genome-wide copy-number analysis by array-based comparative genomic hybridization (array-CGH) identified short somatic duplications that include C-MYB (MYBdup, n = 13 cases of 84 T-ALL, 15%). Expression analysis, including allele-specific approaches, showed stronger C-MYB expression in the MYB-rearranged cases compared with other T-ALLs, and a dramatically skewed C-MYB allele expression in the TCRB-MYB cases, which suggests that a translocation-driven deregulated expression may overcome a cellular attempt to down-regulate C-MYB. Strikingly, profiling of the T-ALLs by clinical, genomic, and large-scale gene expression analyses shows that the TCRB-MYB translocation defines a new T-ALL subtype associated with a very young age for T-cell leukemia (median, 2.2 years) and with a proliferation/mitosis expression signature. By contrast, the MYBdup alteration was associated with the previously defined T-ALL subtypes.