Protein Kinase C Epsilon Overexpression Is Associated With Poor Patient Outcomes in AML and Promotes Daunorubicin Resistance Through p-Glycoprotein-Mediated Drug Efflux.

The protein kinase C (PKC) family of serine/threonine kinases are pleiotropic signaling regulators and are implicated in hematopoietic signaling and development. Only one isoform however, PKCϵ, has oncogenic properties in solid cancers where it is associated with poor outcomes. Here we show that PKCϵ protein is significantly overexpressed in acute myeloid leukemia (AML; 37% of patients). In addition, PKCϵ expression in AML was associated with a significant reduction in complete remission induction and disease-free survival. Examination of the functional consequences of PKCϵ overexpression in normal human hematopoiesis, showed that PKCϵ promotes myeloid differentiation, particularly of the monocytic lineage, and decreased colony formation, suggesting that PKCϵ does not act as an oncogene in hematopoietic cells. Rather, in AML cell lines, PKCϵ overexpression selectively conferred resistance to the chemotherapeutic agent, daunorubicin, by reducing intracellular concentrations of this agent. Mechanistic analysis showed that PKCϵ promoted the expression of the efflux pump, P-GP (ABCB1), and that drug efflux mediated by this transporter fully accounted for the daunorubicin resistance associated with PKCϵ overexpression. Analysis of AML patient samples also showed a link between PKCϵ and P-GP protein expression suggesting that PKCϵ expression drives treatment resistance in AML by upregulating P-GP expression.

Increased expression of RUNX3 inhibits normal human myeloid development.

RUNX3 is a transcription factor dysregulated in acute myeloid leukemia (AML). However, its role in normal myeloid development and leukemia is poorly understood. Here we investigate RUNX3 expression in both settings and the impact of its dysregulation on myelopoiesis. We found that RUNX3 mRNA expression was stable during hematopoiesis but decreased with granulocytic differentiation. In AML, RUNX3 mRNA was overexpressed in many disease subtypes, but downregulated in AML with core binding factor abnormalities, such as RUNX1::ETO. Overexpression of RUNX3 in human hematopoietic stem and progenitor cells (HSPC) inhibited myeloid differentiation, particularly of the granulocytic lineage. Proliferation and myeloid colony formation were also inhibited. Conversely, RUNX3 knockdown did not impact the myeloid growth and development of human HSPC. Overexpression of RUNX3 in the context of RUNX1::ETO did not rescue the RUNX1::ETO-mediated block in differentiation. RNA-sequencing showed that RUNX3 overexpression downregulates key developmental genes, such as KIT and RUNX1, while upregulating lymphoid genes, such as KLRB1 and TBX21. Overall, these data show that increased RUNX3 expression observed in AML could contribute to the developmental arrest characteristic of this disease, possibly by driving a competing transcriptional program favoring a lymphoid fate.

PLATOX: Integrated In Vitro/In Vivo Approach for Screening of Adverse Lung Effects of Graphene-Related 2D Nanomaterials.

Graphene-related two-dimensional nanomaterials possess very technically promising characteristics, but gaps exist regarding their potential adverse health effects. Based on their nano-thickness and lateral micron dimensions, nanoplates exhibit particular aerodynamic properties, including respirability. To develop a lung-focused, in vitro/in vivo screening approach for toxicological hazard assessment, various graphene-related nanoplates, i.e., single-layer graphene (SLG), graphene nanoplatelets (GNP), carboxyl graphene, graphene oxide, graphite oxide and Printex 90® (particle reference) were used. Material characterization preceded in vitro (geno)toxicity screening (membrane integrity, metabolic activity, proliferation, DNA damage) with primary rat alveolar macrophages (AM), MRC-5 lung fibroblasts, NR8383 and RAW 264.7 cells. Submerse cell exposure and material-adapted methods indicated material-, cell type-, concentration-, and time-specific effects. SLG and GNP were finally chosen as in vitro biologically active or more inert graphene showed eosinophils in lavage fluid for SLG but not GNP. The subsequent 28-day inhalation study (OECD 412) confirmed a toxic, genotoxic and pro-inflammatory potential for SLG at 3.2 mg/m3 with an in vivo-ranking of lung toxicity: SLG > GNP > Printex 90®. The in vivo ranking finally pointed to AM (lactate dehydrogenase release, DNA damage) as the most predictive in vitro model for the (geno)toxicity screening of graphene nanoplates.

RUNX3 overexpression inhibits normal human erythroid development.

RUNX proteins belong to a family of transcription factors essential for cellular proliferation, differentiation, and apoptosis with emerging data implicating RUNX3 in haematopoiesis and haematological malignancies. Here we show that RUNX3 plays an important regulatory role in normal human erythropoiesis. The impact of altering RUNX3 expression on erythropoiesis was determined by transducing human CD34+ cells with RUNX3 overexpression or shRNA knockdown vectors. Analysis of RUNX3 mRNA expression showed that RUNX3 levels decreased during erythropoiesis. Functionally, RUNX3 overexpression had a modest impact on early erythroid growth and development. However, in late-stage erythroid development, RUNX3 promoted growth and inhibited terminal differentiation with RUNX3 overexpressing cells exhibiting lower expression of glycophorin A, greater cell size and less differentiated morphology. These results suggest that suppression of RUNX3 is required for normal erythropoiesis. Overexpression of RUNX3 increased colony formation in liquid culture whilst, corresponding RUNX3 knockdown suppressed colony formation but otherwise had little impact. This study demonstrates that the downregulation of RUNX3 observed in normal human erythropoiesis is important in promoting the terminal stages of erythroid development and may further our understanding of the role of this transcription factor in haematological malignancies.

Gata2 as a Crucial Regulator of Stem Cells in Adult Hematopoiesis and Acute Myeloid Leukemia.

Subversion of transcription factor (TF) activity in hematopoietic stem/progenitor cells (HSPCs) leads to the development of therapy-resistant leukemic stem cells (LSCs) that drive fulminant acute myeloid leukemia (AML). Using a conditional mouse model where zinc-finger TF Gata2 was deleted specifically in hematopoietic cells, we show that knockout of Gata2 leads to rapid and complete cell-autonomous loss of adult hematopoietic stem cells. By using short hairpin RNAi to target GATA2, we also identify a requirement for GATA2 in human HSPCs. In Meis1a/Hoxa9-driven AML, deletion of Gata2 impedes maintenance and self-renewal of LSCs. Ablation of Gata2 enforces an LSC-specific program of enhanced apoptosis, exemplified by attenuation of anti-apoptotic factor BCL2, and re-instigation of myeloid differentiation--which is characteristically blocked in AML. Thus, GATA2 acts as a critical regulator of normal and leukemic stem cells and mediates transcriptional networks that may be exploited therapeutically to target key facets of LSC behavior in AML.

Cytotoxic effect of the serotonergic drug 1-(1-Naphthyl)piperazine against melanoma cells.

1-(1-Naphthyl)piperazine (1-NPZ) is a serotonergic derivative of quipazine acting both as antagonist and agonist of different serotonin receptors, with promising results for the management of skin cancer. In this work, we studied the effect of 1-NPZ on human MNT-1 melanoma cells by evaluating its effects on cell viability, ability to form colonies, cell cycle dynamics, reactive oxygen species (ROS) production and apoptosis. Treatment of MNT-1 cells with 1-NPZ for 24h decreased cell viability and induced apoptosis in a dose-dependent manner. Activity against melanoma was confirmed with a different melanoma cell line, SK-MEL-28. Simultaneously, 1-NPZ affected cell cycle progression by mediating a S-phase delay. Higher levels of ROS were also detected in MNT-1 cells after treatment with 1-NPZ. Furthermore, 1-NPZ significantly increased the expression of cyclooxygenase-2 in MNT-1 cells. These findings suggest that 1-NPZ pretreatment is able to induce oxidative stress, and consequently apoptotic cell death in melanoma cells. In conclusion, this study demonstrates the cytotoxic and genotoxic potential of 1-NPZ against melanoma cells.

Development and characterization of novel 1-(1-Naphthyl)piperazine-loaded lipid vesicles for prevention of UV-induced skin inflammation.

1-(1-Naphthyl)piperazine (1-NPZ) has shown promising effects by inhibiting UV radiation-induced immunosuppression. Ultradeformable vesicles are recent advantageous systems capable of improving the (trans)dermal drug delivery. The aim of this study was to investigate 1-NPZ-loaded transethosomes (NPZ-TE) and 1-NPZ-loaded vesicles containing dimethyl sulfoxide (NPZ-DM) as novel delivery nanosystems, and to uncover their chemopreventive effect against UV-induced acute inflammation. Their physicochemical properties were evaluated as follows: vesicles size and zeta potential by dynamic and electrophoretic light scattering, respectively; vesicle deformability by pressure driven transport; rheological behavior by measuring viscosity and I-NPZ entrapment yield by HPLC. In vitro topical delivery studies were performed in order to evaluate the permeation profile of both formulations, whereas in vivo studies sought to assess the photoprotective effect of the selected formulation on irradiated hairless mice by measuring myeloperoxidase activity and the secretion of proinflammatory cytokines. Either NPZ-TE or NPZ-DM exhibited positive results in terms of physicochemical properties. In vitro data revealed an improved permeation of 1-NPZ across pig ear skin, especially by NPZ-DM. In vivo studies demonstrated that NPZ-DM exposure was capable of preventing UVB-induced inflammation and blocking mediators of inflammation in mouse skin. The successful results here obtained encourage us to continue these studies for the management of inflammatory skin conditions that may lead to the development of skin cancers.

Prevention of Photocarcinogenesis by Agonists of 5-HT1A and Antagonists of 5-HT2A Receptors.

Exposure to UV radiation is the principal cause of nonmelanoma skin cancer, a process in which serotonin (5-HT) is intimately involved. This review focuses on the potential of serotonin receptors, namely 5-HT1/2A, as therapeutic targets for prevention of photocarcinogenesis. UV-induced immunosuppression is triggered by a cascade of events initiated when cis-urocanic acid, a UV photoreceptor present in the skin, binds to the serotonin receptor. Serotonin receptor antagonists will therefore attempt to block this association, and in turn, prevent skin cancer induction. In addition, 5-HT2A receptor antagonists are also capable of regulating DNA repair, including the acceleration of nucleotide excision repair. At the same time, UV-induced formation of reactive oxygen species is also reduced by these agents. Since the involvement of serotonin in photocarcinogenesis process is somewhat underexplored as a pertinent therapeutic effect, this review intends to reveal the use of serotonergic drugs as an important strategy to prevent and/or inhibit photocarcinogenesis. Considering the emergency of developing novel therapeutic strategies for skin cancer management, the use of these agents, whose benefits have partially been studied, may be crucial especially if topically applied. Topical nanoformulations containing serotonin receptor agonists and/or antagonists also represent a pioneer concept in this area. Graphical Abstract ᅟ.