MicroRNAs: new players in acute myeloid leukaemia.

MicroRNAs (miRNAs) are short non-coding RNAs that have key functions in a wide array of critical cell processes, including haematopoiesis by regulating the expression of multiple genes. Aberrant miRNA expression has been described in acute myeloid leukaemia suggesting a role in leukaemogenesis. In this review we summarise the current knowledge.

MicroRNA gene expression during retinoic acid-induced differentiation of human acute promyelocytic leukemia.

MicroRNAs (miRNAs) are small non-coding RNAs of 19-25 nucleotides that are involved in the regulation of critical cell processes such as apoptosis, cell proliferation and differentiation. However, little is known about the role of miRNAs in granulopoiesis. Here, we report the expression of miRNAs in acute promyelocytic leukemia patients and cell lines during all-trans-retinoic acid (ATRA) treatment by using a miRNA microarrays platform and quantitative real time-polymerase chain reaction (qRT-PCR). We found upregulation of miR-15a, miR-15b, miR-16-1, let-7a-3, let-7c, let-7d, miR-223, miR-342 and miR-107, whereas miR-181b was downregulated. Among the upregulated miRNAs, miR-107 is predicted to target NFI-A, a gene that has been involved in a regulatory loop involving miR-223 and C/EBPa during granulocytic differentiation. Indeed, we have confirmed that miR-107 targets NF1-A. To get insights about ATRA regulation of miRNAs, we searched for ATRA-modulated transcription factors binding sites in the upstream genomic region of the let-7a-3/let-7b cluster and identified several putative nuclear factor-kappa B (NF-kappaB) consensus elements. The use of reporter gene assays, chromatin immunoprecipitation and site-directed mutagenesis revealed that one proximal NF-kappaB binding site is essential for the transactivation of the let-7a-3/let-7b cluster. Finally, we show that ATRA downregulation of RAS and Bcl2 correlate with the activation of known miRNA regulators of those proteins, let-7a and miR-15a/miR-16-1, respectively.

Multiple silencer elements are involved in regulating the chicken vimentin gene.

Vimentin, a member of the intermediate filament protein family, exhibits tissue- as well as development-specific expression. Transcription factors that are involved in expression of the chicken vimentin gene have been described and include a cis-acting silencer element (SE3) that is involved in the down-regulation of this gene (F. X. Farrell, C. M. Sax, and Z. E. Zehner, Mol. Cell. Biol. 10:2349-2358, 1990). In this study, we report the identification of two additional silencer elements (SE1 and SE2). We show by transfection analysis that all three silencer elements are functionally active and that optimal silencing occurs when multiple (at least two) silencer elements are present. In addition, the previously identified SE3 can be divided into three subregions, each of which is moderately active alone. By gel mobility shift assays, all three silencer elements plus SE3 subregions bind a protein which by Southwestern (DNA-protein) blot analysis is identical in molecular mass (approximately 95 kDa). DNase I footprinting experiments indicate that this protein binds to purine-rich sites. Therefore, multiple elements appear to be involved in the negative regulation of the chicken vimentin gene, which may be important in the regulation of other genes as well.

A negative regulatory factor is missing in a human metastatic breast cancer cell line.

The intermediate filament protein, vimentin, is differentially expressed in various tissues and stages of development and in metastatic versus nonmetastatic breast cancer cell lines. Previously, we have shown vimentin expression to be regulated at least in part by a silencer element which binds a M(r) 95,000 protein and an overriding, antisilencer element which binds a M(r) 140,000 protein. Southwestern blot (DNA-protein) analyses indicate that silencer protein binding activity is missing in the metastatic breast cancer cell line (MDA-MB-231), where vimentin is highly expressed, but is present in the nonmetastatic breast cancer cell line, MCF-7, where vimentin is not expressed. This suggests that the absence of a functional silencer protein may lead to expression of vimentin as well as other genes which contribute to the metastatic state.

The dual epigenetic role of PRMT5 in acute myeloid leukemia: gene activation and repression via histone arginine methylation.

Changes in the enzymatic activity of protein arginine methyltransferase (PRMT) 5 have been associated with cancer; however, the protein's role in acute myeloid leukemia (AML) has not been fully evaluated. Here, we show that increased PRMT5 activity enhanced AML growth in vitro and in vivo while PRMT5 downregulation reduced it. In AML cells, PRMT5 interacted with Sp1 in a transcription repressor complex and silenced miR-29b preferentially via dimethylation of histone 4 arginine residue H4R3. As Sp1 is also a bona fide target of miR-29b, the miR silencing resulted in increased Sp1. This event in turn led to transcription activation of FLT3, a gene that encodes a receptor tyrosine kinase. Inhibition of PRMT5 via sh/siRNA or a first-in-class small-molecule inhibitor (HLCL-61) resulted in significantly increased expression of miR-29b and consequent suppression of Sp1 and FLT3 in AML cells. As a result, significant antileukemic activity was achieved. Collectively, our data support a novel leukemogenic mechanism in AML where PRMT5 mediates both silencing and transcription of genes that participate in a 'yin-yang' functional network supporting leukemia growth. As FLT3 is often mutated in AML and pharmacologic inhibition of PRMT5 appears feasible, the PRMT5-miR-29b-FLT3 network should be further explored as a novel therapeutic target for AML.

Next-generation XPO1 inhibitor shows improved efficacy and in vivo tolerability in hematological malignancies.

The nuclear export receptor, Exportin 1 (XPO1), mediates transport of growth-regulatory proteins, including tumor suppressors, and is overactive in many cancers, including chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML) and aggressive lymphomas. Oral selective inhibitor of nuclear export (SINE) compounds that block XPO1 function were recently identified and hold promise as a new therapeutic paradigm in many neoplasms. One of these compounds, KPT-330 (selinexor), has made progress in Phase I/II clinical trials, but systemic toxicities limit its administration to twice-per-week and requiring supportive care. We designed a new generation SINE compound, KPT-8602, with a similar mechanism of XPO1 inhibition and potency but considerably improved tolerability. Efficacy of KPT-8602 was evaluated in preclinical animal models of hematological malignancies, including CLL and AML. KPT-8602 shows similar in vitro potency compared with KPT-330 but lower central nervous system penetration, which resulted in enhanced tolerability, even when dosed daily, and improved survival in CLL and AML murine models compared with KPT-330. KPT-8602 is a promising compound for further development in hematological malignancies and other cancers in which upregulation of XPO1 is seen. The wider therapeutic window of KPT-8602 may also allow increased on-target efficacy leading to even more efficacious combinations with other targeted anticancer therapies.

Pharmacological targeting of miR-155 via the NEDD8-activating enzyme inhibitor MLN4924 (Pevonedistat) in FLT3-ITD acute myeloid leukemia.

High levels of microRNA-155 (miR-155) are associated with poor outcome in acute myeloid leukemia (AML). In AML, miR-155 is regulated by NF-κB, the activity of which is, in part, controlled by the NEDD8-dependent ubiquitin ligases. We demonstrate that MLN4924, an inhibitor of NEDD8-activating enzyme presently being evaluated in clinical trials, decreases binding of NF-κB to the miR-155 promoter and downregulates miR-155 in AML cells. This results in the upregulation of the miR-155 targets SHIP1, an inhibitor of the PI3K/Akt pathway, and PU.1, a transcription factor important for myeloid differentiation, leading to monocytic differentiation and apoptosis. Consistent with these results, overexpression of miR-155 diminishes MLN4924-induced antileukemic effects. In vivo, MLN4924 reduces miR-155 expression and prolongs the survival of mice engrafted with leukemic cells. Our study demonstrates the potential of miR-155 as a novel therapeutic target in AML via pharmacologic interference with NF-κB-dependent regulatory mechanisms. We show the targeting of this oncogenic microRNA with MLN4924, a compound presently being evaluated in clinical trials in AML. As high miR-155 levels have been consistently associated with aggressive clinical phenotypes, our work opens new avenues for microRNA-targeting therapeutic approaches to leukemia and cancer patients.

Targeting leukemia stem cells in vivo with antagomiR-126 nanoparticles in acute myeloid leukemia.

Current treatments for acute myeloid leukemia (AML) are designed to target rapidly dividing blast populations with limited success in eradicating the functionally distinct leukemia stem cell (LSC) population, which is postulated to be responsible for disease resistance and relapse. We have previously reported high miR-126 expression levels to be associated with a LSC-gene expression profile. Therefore, we hypothesized that miR-126 contributes to 'stemness' and is a viable target for eliminating the LSC in AML. Here we first validate the clinical relevance of miR-126 expression in AML by showing that higher expression of this microRNA (miR) is associated with worse outcome in a large cohort of older (⩾60 years) cytogenetically normal AML patients treated with conventional chemotherapy. We then show that miR-126 overexpression characterizes AML LSC-enriched cell subpopulations and contributes to LSC long-term maintenance and self-renewal. Finally, we demonstrate the feasibility of therapeutic targeting of miR-126 in LSCs with novel targeting nanoparticles containing antagomiR-126 resulting in in vivo reduction of LSCs likely by depletion of the quiescent cell subpopulation. Our findings suggest that by targeting a single miR, that is, miR-126, it is possible to interfere with LSC activity, thereby opening potentially novel therapeutic approaches to treat AML patients.

[Postoperative gallbladder and systemic candidiasis]. / Candidiasis vesicular y sistémica post-operatoria.

Gangrenous cholecystitis occurred in an immunodepressed patient with generalized neoplasia. Evolution was rapid and severe. Emergency cholecystectomy and systemic treatment with Anphotherycin were life-saving.

Myelodysplastic syndromes: a community hospital-based study of prognostic factors and International Prognostic Scoring System.

Various prognostic factors and the International Prognosis Scoring System (IPSS) were assessed in our community hospital-based retrospective study of 55 cases of myelodysplastic syndromes (MDS). All cases were reviewed for clinical, hematologic, histopathologic, and cytogenetic data. The median follow-up was 1.61 years. Twenty patients (36%) were classified as refractory anemia (RA); seven (13%) as refractory anemia with ringed sideroblasts (RARS); 13 (24%) as chronic myelomonocytic leukemia (CMML); 11 (20%) as refractory anemia with excess blasts (RAEB); and four (7%) as refractory anemia with excess blasts in transformation (RAEB-t). Twenty-seven (49%) died during the follow-up period, seven with acute myelogenous leukemia (AML). The median survival was 2.8 years. The variables that showed association with survival by univariate analysis included the absolute neutrophil count, French-American-British (FAB) subtype, percentage of blasts, number of cytopenias, abnormal localization of immature precursors, and IPSS score. When entered into a regression model, IPSS showed a trend towards an association with survival (P 0.09). We conclude that the IPSS can prognostically stratify MDS patients. However, no independent prognostic factor was confirmed in our analysis. Further studies are needed to assess the utility of IPSS.

[Acute eosinophilic pneumonia. Case report and review of the literature]. / Neumonía eosinofílica aguda. Presentación de un caso y revisión de la literatura.

A 37 year old female smoker was admitted with an acute episode of fever and pulmonary infiltrates followed by respiratory failure requiring mechanical ventilation in less than 24 hours. After empiric antibiotic therapy fiberoptic bronchoscopy and bronchoalveolar lavage (BAL) were carried out. Abnormal findings were limited to highly increased eosinophilic count in BAL. Blood eosinophils were normal. Acute eosinophilic pneumonia was diagnosed and methyl prednisolone was administered; 48 hours afterwards, infiltrates had partially resolved and the patient was weaned from ventilator. After two years follow up, she has not had recurrences of the episode, is asymptomatic and her chest Xray is normal. A review of the literature is presented and physiopathological mechanisms for acute eosinophilic pneumonia are considered. Acute eosinophilic pneumonia is a rare but potentially curable cause of respiratory failure that should be included in diagnostic considerations.

[Prototype of light source for photodynamic therapy in Centre of Electron Microscopy]. / Prototipo de fuente lumínica para terapia fotodinámica diseñado en el Centro de Microscopía Electrónica.

The Photodynamic Therapy (PDT) is a cancer treatment based on specific accumulation of a photosensitizer in the malignant tissue and its subsequent irradiation at appropriate wave lengths induce the production of oxygen singlets responsible for peroxidation of cell organelles and cell death. A prototype of a 630 nm non coherent light source designed and constructed at the Centre of Electron Microscopy of The Cordoba National University allowed a successful application of PDT, in nonmelanoma skin tumors, for the first time in this Country. A topical treatment with 20% delta aminolevulinic acid (ALA) in aqueous solution was applied in 100 lesions of actinic keratosis of 27 patients. The results obtained in this study were the following: Complete Remission (RC) 84%, Partial Remission (RP) 10%, No Response (SR) 0% and No data (SD) 6%. In the latter group are included those patients who did not return for reevaluation. The high efficiency plus the excellent cosmetic response and low aggressiveness, make the PDT the method of choice treatment of this skin pathology. In addition to be no invasive and well tolerated, the prototype of light source used in this study, was remarkably effective.

[Marjolin ulcer]. / Ulcera de Marjolin.

Marjolin's ulcers can produce neoplasms. They appear on previous scars produced by old three degree burns. Sometimes they appear after 50 years of the previous accident. Three patients showing squamous cell epitheliomas are presented, and the opportunity and technique of the surgical treatment is discussed.

Increased anti-leukemic activity of decitabine via AR-42-induced upregulation of miR-29b: a novel epigenetic-targeting approach in acute myeloid leukemia.

Histone deacetylase (HDAC) inhibitors either alone or in combination with hypomethylating agents have limited clinical effect in acute myeloid leukemia (AML). Previously, we demonstrated that AML patients with higher miR (microRNA)-29b expression had better response to the hypomethylating agent decitabine. Therefore, an increase in miR-29b expression preceding decitabine treatment may provide a therapeutic advantage. We previously showed that miR-29b expression is suppressed by a repressor complex that includes HDACs. Thus, HDAC inhibition may increase miR-29b expression. We hypothesized that priming AML cells with the novel HDAC inhibitor (HDACI) AR-42 would result in increased response to decitabine treatment via upregulation of miR-29b. Here, we show that AR-42 is a potent HDACI in AML, increasing miR-29b levels and leading to downregulation of known miR-29b targets (that is, SP1, DNMT1, DNMT3A and DNMT3B). We then demonstrated that the sequential administration of AR-42 followed by decitabine resulted in a stronger anti-leukemic activity in vitro and in vivo than decitabine followed by AR-42 or either drug alone. These preclinical results with AR-42 priming before decitabine administration represent a promising, novel treatment approach and a paradigm shift with regard to the combination of epigenetic-targeting compounds in AML, where decitabine has been traditionally given before HDACIs.

The association of statins plus LDL receptor-targeted liposome-encapsulated doxorubicin increases in vitro drug delivery across blood-brain barrier cells.

BACKGROUND AND PURPOSE: The passage of drugs across the blood-brain barrier (BBB) limits the efficacy of chemotherapy in brain tumours. For instance, the anticancer drug doxorubicin, which is effective against glioblastoma in vitro, has poor efficacy in vivo, because it is extruded by P-glycoprotein (Pgp/ABCB1), multidrug resistance-related proteins and breast cancer resistance protein (BCRP/ABCG2) in BBB cells. The aim of this study was to convert poorly permeant drugs like doxorubicin into drugs able to cross the BBB. EXPERIMENTAL APPROACH: Experiments were performed on primary human cerebral microvascular endothelial hCMEC/D3 cells, alone and co-cultured with human brain and epithelial tumour cells. KEY RESULTS: Statins reduced the efflux activity of Pgp/ABCB1 and BCRP/ABCG2 in hCMEC/D3 cells by increasing the synthesis of NO, which elicits the nitration of critical tyrosine residues on these transporters. Statins also increased the number of low-density lipoprotein (LDL) receptors exposed on the surface of BBB cells, as well as on tumour cells like human glioblastoma. We showed that the association of statins plus drug-loaded nanoparticles engineered as LDLs was effective as a vehicle for non-permeant drugs like doxorubicin to cross the BBB, allowing its delivery into primary and metastatic brain tumour cells and to achieve significant anti-tumour cytotoxicity. CONCLUSIONS AND IMPLICATIONS: We suggest that our 'Trojan horse' approach, based on the administration of statins plus a LDL receptor-targeted liposomal drug, might have potential applications in the pharmacological therapy of different brain diseases for which the BBB represents an obstacle.

Pulmonary Nocardia and Aspergillus co-infection in a patient with chronic graft-versus-host disease.

Immunosuppression and chronic graft-versus-host disease (GVHD) are major risk factors for the development of invasive pulmonary aspergillosis in bone marrow transplant patients. Although nocardial infections are well described in hematopoietic stem cell transplantation (HSCT) recipients, little information is available about the incidence of nocardiosis in patients with chronic GVHD after HSCT. Coexistence of invasive pulmonary aspergillosis and nocardiosis following non-myeloablative HSCT has not been reported previously. With the increasing use of pentostatin in the treatment of chronic GVHD in future and other nucleoside analogues as preparative regimens in patients undergoing reduced-intensity conditioning transplantation, the possibility of co-infection with rare organisms should be kept in mind while assessing at-risk patients.

MicroRNAs and noncoding RNAs in hematological malignancies: molecular, clinical and therapeutic implications.

MicroRNAs (miRNAs) are a family of 19-24 nucleotide noncoding RNAs (ncRNAs) with posttranscriptional regulatory functions. Increasing evidences from the literature show that miRNAs play a pivotal role in human tumorigenesis. Many studies have addressed the role of miRNAs in normal hematopoiesis, giving an interpretative key to the aberrancies of expression observed in human hematological malignancies. Moreover, the recent demonstration that other ncRNAs, the ultraconserved genes (UCGs) or transcribed ultraconserved regions (T-UCRs), are involved in human cancerogenesis, suggests that the wider family of ncRNAs (including both miRNAs and UCGs) could contribute to the development of the malignant phenotype. Here we review the main studies investigating the role of miRNAs and UCRs in both normal hemopoiesis and hematological malignancies, and identify the molecular, clinical and therapeutic implications of these recent findings.