Guilherme A. dos Santos

Increased expression of miR-221 is associated with shorter overall survival in T-cell acute lymphoid leukemia.

BackgroundCD56 expression has been associated with a poor prognosis in lymphoid neoplasms, including T-cell acute lymphoblastic leukemia (T-ALL). MicroRNAs (miRNAs) play an important role in lymphoid differentiation, and aberrant miRNA expression has been associated with treatment outcome in lymphoid malignancies. Here, we evaluated miRNA expression profiles in normal thymocytes, mature T-cells, and T-ALL samples with and without CD56 expression and correlated microRNA expression with treatment outcome.MethodsThe gene expression profile of 164 miRNAs were compared for T-ALL/CD56+ (n=12) and T-ALL/CD56- (n=36) patients by Real-Time Quantitative PCR. Based on this analysis, we decided to evaluate miR-221 and miR-374 expression in individual leukemic and normal samples.ResultsmiR-221 and miR-374 were expressed at significantly higher levels in T-ALL/CD56+ than in T-ALL/CD56- cells and in leukemic blasts compared with normal thymocytes and peripheral blood (PB) T-cells. Age at diagnosis (15 or less vs grater than 15 years; HR: 2.19, 95% CI: 0.98-4.85; P=0.05), miR-221 expression level (median value as cut off in leukemic samples; HR: 3.17, 95% CI: 1.45-6.92; P=0.004), and the expression of CD56 (CD56-vs CD56+; HR: 2.99, 95% CI: 1.37-6.51; P=0.006) were predictive factors for shorter overall survival; whereas, only CD56 expression (HR: 2.73, 95% CI: 1.03-7.18; P=0.041) was associated with a shorter disease-free survival rate.ConclusionsmiR-221 is highly expressed in T-ALL and its expression level may be associated with a poorer prognosis.

Linker for Activation of T-cell Family Member2 (LAT2) a Lipid Raft Adaptor Protein for AKT Signaling, Is an Early Mediator of Alkylphospholipid Anti-leukemic Activity

Lipid rafts are highly ordered membrane domains rich in cholesterol and sphingolipids that provide a scaffold for signal transduction proteins; altered raft structure has also been implicated in cancer progression. We have shown that 25 μm 10-(octyloxy) decyl-2-(trimethylammonium) ethyl phosphate (ODPC), an alkylphospholipid, targets high cholesterol domains in model membranes and induces apoptosis in leukemia cells but spares normal hematopoietic and epithelial cells under the same conditions. We performed a quantitative (SILAC) proteomic screening of ODPC targets in a lipid-raft-enriched fraction of leukemic cells to identify early events prior to the initiation of apoptosis. Six proteins, three with demonstrated palmitoylation sites, were reduced in abundance. One, the linker for activation of T-cell family member 2 (LAT2), is an adaptor protein associated with lipid rafts in its palmitoylated form and is specifically expressed in B lymphocytes and myeloid cells. Interestingly, LAT2 is not expressed in K562, a cell line more resistant to ODPC-induced apoptosis. There was an early loss of LAT2 in the lipid-raft-enriched fraction of NB4 cells within 3 h following treatment with 25 μm ODPC. Subsequent degradation of LAT2 by proteasomes was observed. Twenty-five μm ODPC inhibited AKT activation via myeloid growth factors, and LAT2 knockdown in NB4 cells by shRNA reproduced this effect. LAT2 knockdown in NB4 cells also decreased cell proliferation and increased cell sensitivity to ODPC (7.5×), perifosine (3×), and arsenic trioxide (8.5×). Taken together, these data indicate that LAT2 is an early mediator of the anti-leukemic activity of alkylphospholipids and arsenic trioxide. Thus, LAT2 may be used as a target for the design of drugs for cancer therapy.

The expression of ΔNTP73, TATP73 and TP53 genes in acute myeloid leukaemia is associated with recurrent cytogenetic abnormalities and in vitro susceptibility to cytarabine cytotoxicity

TP73 encodes for two proteins: full‐length TAp73 and ΔNp73, which have little transcriptional activity and exert dominant‐negative function towards TP53 and TAp73. We compared TATP73 and ΔNTP73 expression in acute myeloid leukaemia (AML) samples and normal CD34+ progenitors. Both forms were more highly expressed in leukaemic cells. Amongst AML blasts, TATP73 was more expressed in AML harbouring the recurrent genetic abnormalities (RGA): PML‐RARA, RUNX1‐RUNX1T1 and CBFB‐MYH11, whereas higher ΔNTP73 expression was detected in non‐RGA cases. TP53 expression did not vary according to ΔNTP73/TATP73 expression ratio. Leukaemic cells with higher ΔNTP73/TATP73 ratios were significantly more resistant to cytarabine‐induced apoptosis.

Results of FLT3 mutation screening and correlations with immunophenotyping in 169 Brazilian patients with acute myeloid leukemia

A. R. Lucena-Araujo :D. L. Souza : F. M. de Oliveira : M. T. L. Benicio : L. L. Figueiredo-Pontes : B. A. Santana-Lemos :G. A. dos Santos : R. H. Jacomo : E. M. Rego (*) Hematology Division, Department of Internal Medicine, National Institute of Science and Technology on Cell Based Therapy, Medical School of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, Brazil e-mail: emrego@hcrp.fmrp.usp.br

Apoptosis induction by (+)α-tocopheryl succinate in the absence or presence of all-trans retinoic acid and arsenic trioxide in NB4, NB4-R2 and primary APL cells

We analyzed the effect of (+)alpha-tocopheryl succinate (alpha-TOS) alone or associated with arsenic trioxide (ATO) or all-trans retinoid acid (ATRA) in acute promyelocytic leukemia (APL). alpha-TOS-induced apoptosis in APL clinical samples and in ATRA-sensitive (NB4) and ATRA-resistant (NB4-R2) APL cell lines. The effective dose 50% (ED-50) was calculated to be 71 and 58muM, for NB4 and NB4-R2, respectively. alpha-TOS neither induced nor modified ATRA-induced differentiation of APL cells, and did not affect the proliferation and differentiation of normal CD34(+) hematopoietic progenitors in methylcellulose assays. alpha-TOS exerted a moderate antagonistic effect to ATO-induced apoptosis when treatment was done simultaneously but when alpha-TOS was added 24h after ATO, an additive effect was observed. Our results support the concept of alpha-TOS as an anti-leukemic compound which spares normal hematopoiesis.

Characterization of the stimulus for reactive oxygen species generation in calcium-overloaded mitochondria

Abstract We have used two different probes with distinct detection properties, dichlorodihydrofluorescein diacetate and Amplex Red/horseradish peroxidase, as well as different respiratory substrates and electron transport chain inhibitors, to characterize the reactive oxygen species (ROS) generation by the respiratory chain in calcium-overloaded mitochondria. Regardless of the respiratory substrate, calcium stimulated the mitochondrial generation of ROS, which were released at both the mitochondrial-matrix side and the extra-mitochondrial space, in a way insensitive to the mitochondrial permeability transition pores inhibitor cyclosporine A. In glutamate/malate-energized mitochondria, inhibition at complex I or complex III (ubiquinone cycle) similarly modulated ROS generation at either mitochondrial-matrix side or extra-mitochondrial space; this also occurred when the backflow of electrons to complex I in succinate-energized mitochondria was inhibited. On the other hand, in succinate-energized mitochondria the modulation of ROS generation at mitochondrial-matrix side or extra-mitochondrial space depends on the site of complex III which was inhibited. These results allow a straight comparison between the effects of different respiratory substrates and electron transport chain inhibitors on ROS generation at either mitochondrial-matrix side or extra-mitochondrial space in calcium-overloaded mitochondria.

Abstract A229: Mechanism of action of perifosine on the mantle cell lymphoma line, Granta-519.

Lipid rafts are highly ordered membrane domains that are enriched in cholesterol and sphingolipids and provides a scaffold for signal transduction. Altered raft assembly has been implicated in cancer progression. Alkylphospholipids have been used with promising specific cytotoxic effects in different types of cancer cells. These alkylphospholipids act by interaction with cell membranes and protein lipid rafts. Cell viability was determined by trypan blue assay. Cell cycle was evaluated by flow cytometry. Lipid rafts were isolated using sucrose density-gradient centrifugation after 12 hours of treatment with perifosine. Antibody microarray and western blotting was used to determine levels of key cellular proteins in special on cell signaling proteins. We showed that perifosine, an alkylphospholipid, targets raft-like domains in model membranes and induces apoptosis in mantle cell lymphomas (ED-50 20 μM - Granta-519) by activation of both extrinsic and intrinsic pathways. We observed an effect on the organization of the cell cycle, with increase of the population at G2/M phase (8,6% ± 0,6% to 27,4% ± 2,4%). Cyclin D1 decrease was detected at 24 hours of treatment. We also showed that perifosine downregulates NTAL/LAB (Non-T-cell activation linker/ Linker for activation of B-cells), an adaptor protein that is targeted to rafts by palmitoylation and specifically expressed in hematopoietic tissues. Moreover, perifosine induced a loss of NTAL/LAB in the lipid rafts of Granta-519 cells after 12 hours of treatment. Since NTAL/LAB may function as an adaptor protein in AKT signalization, we evaluate the effect of perifosine on the ATK pathway. We showed that perifosine lead to dephosphorylation of AKT and downstream components of AKT signaling. Moreover, in functional experiments, perifosine inhibited AKT activation by CD40L or IL-4 in Granta-519 cells, after few minutes of incubation, in a way similar to the specific PI3K inhibitor wortmannin. Treatment with methyl-β-cyclodextrin, a compound for cholesterol depletion, potentiated this effect. This suggests that NTAL/LAB translation is highly regulated and dependent on a functional AKT pathway. Our results indicate that perifosine acts on NTAL/LAB presumable by interference with its protein-lipid interactions and consequently AKT signaling. Our results demonstrate that a lipid raft targeting drug may present several effects on signal transduction, causing a severe toxicity to mantle cell lymphoma. Moreover, adaptor proteins like NTAL/LAB emerge as possible new therapeutic targets in lymphoma. This research was supported by FAPESP, CNPq and CAPES. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A229. Citation Format: Germano A. Ferreira, Carolina H. Thome, Guilherme A. dos Santos, Priscila S. Scheucher, Andreia M. Leopoldino, Ana M. Simao, Clarice Izume, Rodrigo A. Panepucci, Pietro Ciancaglini, Eduardo M. Rego, Vitor M. Faca, Lewis J. Greene. Mechanism of action of perifosine on the mantle cell lymphoma line, Granta-519. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A229.