Barbara A. Santana-Lemos

Halofuginone has anti-proliferative effects in acute promyelocytic leukemia by modulating the transforming growth factor beta signaling pathway

Promyelocytic leukemia-retinoic acid receptor alpha (PML-RARα) expression in acute promyelocytic leukemia (APL) impairs transforming growth factor beta (TGFβ) signaling, leading to cell growth advantage. Halofuginone (HF), a low-molecular-weight alkaloid that modulates TGFβ signaling, was used to treat APL cell lines and non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice subjected to transplantation with leukemic cells from human chorionic gonadotrophin-PML-RARα transgenic mice (TG). Cell cycle analysis using incorporated bromodeoxyuridine and 7-amino-actinomycin D showed that, in NB4 and NB4-R2 APL cell lines, HF inhibited cellular proliferation (P<0.001) and induced apoptosis (P = 0.002) after a 24-hour incubation. Addition of TGFβ revealed that NB4 cells were resistant to its growth-suppressive effects and that HF induced these effects in the presence or absence of the cytokine. Cell growth inhibition was associated with up-regulation of TGFβ target genes involved in cell cycle regulation (TGFB, TGFBRI, SMAD3, p15, and p21) and down-regulation of MYC. Additionally, TGFβ protein levels were decreased in leukemic TG animals and HF in vivo could restore TGFβ values to normal. To test the in vivo anti-leukemic activity of HF, we transplanted NOD/SCID mice with TG leukemic cells and treated them with HF for 21 days. HF induced partial hematological remission in the peripheral blood, bone marrow, and spleen. Together, these results suggest that HF has anti-proliferative and anti-leukemic effects by reversing the TGFβ blockade in APL. Since loss of the TGFβ response in leukemic cells may be an important second oncogenic hit, modulation of TGFβ signaling may be of therapeutic interest.

Synthetic phosphoethanolamine has in vitro and in vivo anti-leukemia effects

Background:We recently showed that synthetic phosphoethanolamine reduces tumour growth and inhibits lung metastasis in vivo. Here, we investigated its anti-leukaemia effects using acute promyelocytic leukaemia (APL) as a model.Methods:Cytotoxic effects of Pho-s on leukaemia cells were evaluated by MTT assay. Leukaemic cells obtained from hCG-PML-RARa transgenic mice were transplanted to NOD/SCID mice. After the animals were diagnosed as leukaemic, treatment started with Pho-s using all-trans retinoid acid or daunorubicin as positive control or and saline control. Cell morphology and immunophenotyping were used to detect the undifferentiated blast cells in the spleen, liver and bone marrow. The induction of apoptosis in vitro and in malignant leukaemic clones was evaluated.Results:Synthetic phosphoethanolamine is cytotoxic and induces apoptosis through the mitochondrial pathway in vitro to leukaemia cell lines. In vivo Pho-s exhibits anti-proliferative effects in APL model reducing the number of CD117+ and Gr-1+ immature myeloid cells in the BM, spleen and liver. Synthetic phosphoethanolamine impairs the expansion of malignant clones CD34+/CD117+, CD34+ and Gr-1+ in the BM. In addition, Pho-s induces apoptosis of immature cells in the spleen and liver, a notable effect.Conclusion:Synthetic phosphoethanolamine has anti-leukaemic effects in an APL model by inhibiting malignant clone expansion, suggesting that it is an interesting compound for leukaemia treatment.

The association of ICAM-1 Exon 6 (E469K) but not of ICAM-1 Exon 4 (G241R) and PECAM-1 Exon 3 (L125V) polymorphisms with the development of differentiation syndrome in acute promyelocytic leukemia.

The use of all trans‐retinoic acid (ATRA) is the basis of treatment of acute promyelocytic leukemia (APL) and represents the paradigm of differentiation therapy. In general, ATRA is well‐tolerated but may be associated with a potentially lethal side‐effect, referred to as retinoic acid or differentiation syndrome (DS). The cellular and molecular mechanisms of DS are poorly understood and involve changes in the adhesive qualities and cytokine secretion of leukemic cells during ATRA‐induced differentiation. As leukocyte extravasation is a key event in DS pathogenesis, we analyzed the association between the polymorphisms at Exon 4 (G241R) and Exon 6 (E469K) of ICAM‐1 and Exon 3 (L125V) of PECAM‐1 genes with DS development in APL patients treated with ATRA and anthracyclines. DS was diagnosed in 23/127 (18.1%) APL patients at an average of 11.5 days after the start of ATRA. All patients presented respiratory distress associated with increased ground‐glass opacity in chest radiographies. Other accompanying symptoms were: fever not attributable to infection (65.2%), generalized edema (37.5%), weight gain (37.5%), and impairment of renal function (8.6%). We detected an association between development of DS and the AA genotype at Codon 469 of ICAM‐1 (odds ratio of 3.5; 95% confidence interval: 1.2–10.2). Conversely, no significant association was detected between G241R or L125V polymorphisms at Exon 4 of ICAM‐1 and Exon 3 of PECAM‐1, respectively. Our results suggest that susceptibility to DS in APL patients may be influenced by genetic variation in adhesion molecule loci.

The CEBPA gene is down-regulated in acute promyelocytic leukemia and its upstream promoter, but not the core promoter, is highly methylated

Impairment of CCAAT Enhancer Binding Protein alpha (CEBPA) function is a common finding in acute myeloid leukemia; nevertheless, its relevance for acute promyelocytic leukemia pathogenesis is unclear. We analyzed the expression and assessed the methylation status of the core and upstream promoters of CEBPA in acute promyelocytic leukemia at diagnosis. Patients with acute promyelocytic leukemia (n=18) presented lower levels of CEBPA expression compared to healthy controls (n=5), but higher levels than those in acute myeloid leukemia with t(8;21) (n=9) and with inv(16) (n=5). Regarding the core promoter, we detected no methylation in 39 acute promyelocytic leukemia samples or in 8 samples from controls. In contrast, analysis of the upstream promoter showed methylation in 37 of 39 samples, with 17 patients showing methylation levels over 30%. Our results corroborate data obtained in animal models showing that CEBPA is down-regulated in acute promyelocytic leukemia stem cells and suggest that epigenetic mechanisms may be involved.

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.

Antibody-targeted horseradish peroxidase associated with indole-3-acetic acid induces apoptosis in vitro in hematological malignancies

Indole-3-acetic acid (IAA), when oxidized by horseradish peroxidase (HRP), is transformed into cytotoxic molecules capable of inducing cell injury. The aim of this study was to test if, by targeting hematopoietic tumors with HRP-conjugated antibodies in association with IAA treatment, there is induction of apoptosis. We used two lineages of hematologic tumors: NB4, derived from acute promyelocytic leukemia (APL) and Granta-519 from mantle cell lymphoma (MCL). We also tested cells from 12 patients with acute myeloid leukemia (AML) and from 10 patients with chronic lymphocytic leukemia (CLL). HRP targeting was performed with anti-CD33 or anti-CD19 antibodies (depending on the origin of the cell), followed by incubation with goat anti-mouse antibody conjugated with HRP. Eight experimental groups were analyzed: control, HRP targeted, HRP targeted and incubated with 1, 5 and 10mM IAA, and cells not HRP targeted but incubated with 1, 5 and 10mM IAA. Apoptosis was analyzed by flow cytometry using annexin V-FITC and propidium iodide labeling. Results showed that apoptosis was dependent on the dose of IAA utilized, the duration of exposure to the prodrug and the origin of the neoplasia. Targeting HRP with antibodies was efficient in activating IAA and inducing 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

Methionine-induced hyperhomocysteinemia reverts fibrinolytic pathway activation in a murine model of acute promyelocytic leukemia

Increased fibrinolysis is an important component of acute promyelocytic leukemia (APL) bleeding diathesis. APL blasts overexpress annexin II (ANXII), a receptor for tissue plasminogen activator (tPA), and plasminogen, thereby increasing plasmin generation. Previous studies suggested that ANXII plays a pivotal role in APL coagulopathy. ANXII binding to tPA can be inhibited by homocysteine and hyperhomocysteinemia can be induced by L-methionine supplementation. In the present study, we used an APL mouse model to study ANXII function and the effects of hyperhomocysteinemia in vivo. Leukemic cells expressed higher ANXII and tPA plasma levels (11.95 ng/mL in leukemic vs 10.74 ng/mL in wild-type; P = .004). In leukemic mice, administration of L-methionine significantly increased homocysteine levels (49.0 μmol/mL and < 6.0 μmol/mL in the treated and nontreated groups, respectively) and reduced tPA levels to baseline concentrations. The latter were also decreased after infusion of the LCKLSL peptide, a competitor for the ANXII tPA-binding site (11.07 ng/mL; P = .001). We also expressed and purified the p36 component of ANXII in Pichia methanolica. The infusion of p36 in wild-type mice increased tPA and thrombin-antithrombin levels, and the latter was reversed by L-methionine administration. The results of the present study demonstrate the relevance of ANXII in vivo and suggest that methionine-induced hyperhomocysteinemia may reverse hyperfibrinolysis in APL.

A Nonrandomized Trial of Progressive Resistance Training Intervention in Women With Polycystic Ovary Syndrome and Its Implications in Telomere Content.

Background: Physical activity is known to relieve the metabolic complications of polycystic ovary syndrome (PCOS), and exercise is also associated with telomere biology. We investigated the changes induced by progressive resistance training (PRT) in telomere content and metabolic disorder in women with PCOS and controls. Participants and Methods: Forty-five women with PCOS and 52 healthy women aged 18 to 37 years were submitted to PRT. A linear periodization of PRT was prepared based on a trend of decreasing volume and intensity throughout the training period. The volunteers performed PRT 3 times a week for 4 months. The participants’ physical characteristics and hormonal concentrations were measured before and after PRT, as telomere content that was measured using quantitative real-time polymerase chain reaction. Results: Briefly, Progressive resistance training reduced waist circumference, body fat percentage, plasma testosterone and sex hormone-binding globulin concentrations, glycemia, and free androgen index. Fasting insulin and insulin resistance index were greater in women with PCOS. Androstenedione and homocysteine increased after PRT. There were no differences in telomere content between controls (0.96 ± 0.3 before vs 0.85 ± 0.21 after) and women with PCOS (0.94 ± 0.33 before vs 0.88 ± 0.39 after). Adjusted analysis showed telomere shortening after PRT in all women (0.95 ± 0.31 before vs 0.86 ± 0.31 after; P = .03). In women with PCOS, increased homocysteine levels were related to telomere reduction and increased androstenedione was positively correlated with telomere content after PRT. Conclusions: Progressive resistance training had positive effects on the hormonal and physical characteristics of women with PCOS and controls, but telomere content was reduced and homocysteine level increased in all participants.

Acquired TERT promoter mutations stimulate TERT transcription in mantle cell lymphoma

Mantle cell lymphoma (MCL) is an aggressive lymphoid neoplasm with poor prognosis. Acquired telomerase reverse transcriptase gene promoter (TERTp) mutations are among the most frequent somatic non‐coding mutations in cancers. In this study, the prevalence of TERTp mutations in 24 MCL and 21 other lymphoid neoplasias (oLN) was investigated. Eight MCL samples (33%) carried TERTp mutations, two homozygous and six heterozygous (seven C228T and one C250T), which directly correlated with higher TERT transcription, mitochondrial DNA copy number, and IGHV mutational status in MCL neoplastic cells. TERTp mutations were not found in oLN. TERTp mutations correlated with more lymphoma proliferation and tumor burden, as suggested by the higher number of lymphoma cells circulating in peripheral blood, and tended to associate with longer MCL telomeres, especially in homozygous mutants, although not statistically significant. Telomere‐biology genes were overexpressed in MCL cells in comparison to healthy lymphocytes, but were not influenced by mutation status. The findings described for the first time that acquired TERTp mutations are common in MCL but not in other lymphoid neoplasms. It was also demonstrated that TERTp mutations are associated with higher TERT mRNA expression in MCL cells in vivo and higher tumor burden, suggesting these mutations as a driver event in MCL development and progression. Am. J. Hematol. 91:481–485, 2016.