Exploring the Leukemogenic Potential of GATA-1S, the Shorter Isoform of GATA-1: Novel Insights into Mechanisms Hampering Respiratory Chain Complex II Activity and Limiting Oxidative Phosphorylation Efficiency.

GATA-1 is a key regulator of hematopoiesis. A balanced ratio of its two isoforms, GATA-1FL and GATA-1S, contributes to normal hematopoiesis, whereas aberrant expression of GATA-1S alters the differentiation/proliferation potential of hematopoietic precursors and represents a poor prognostic factor in myeloid leukemia. We previously reported that GATA-1S over-expression correlates with high levels of the succinate dehydrogenase subunit C (SDHC). Alternative splicing variants of the SDHC transcript are over-expressed in several tumors and act as potent dominant negative inhibitors of SDH activity. With this in mind, we investigated the levels of SDHC variants and the oxidative mitochondrial metabolism in myeloid leukemia K562 cells over-expressing GATA-1 isoforms. Over-expression of SDHC variants accompanied by decreased SDH complex II activity and oxidative phosphorylation (OXPHOS) efficiency was found associated only with GATA-1S. Given the tumor suppressor role of SDH and the effects of OXPHOS limitations in leukemogenesis, identification of a link between GATA-1S and impaired complex II activity unveils novel pro-leukemic mechanisms triggered by GATA-1S. Abnormal levels of GATA-1S and SDHC variants were also found in an acute myeloid leukemia patient, thus supporting in vitro results. A better understanding of these mechanisms can contribute to identify novel promising therapeutic targets in myeloid leukemia.

Antagonizing S1P3 Receptor with Cell-Penetrating Pepducins in Skeletal Muscle Fibrosis.

S1P is the final product of sphingolipid metabolism, which interacts with five widely expressed GPCRs (S1P1-5). Increasing numbers of studies have indicated the importance of S1P3 in various pathophysiological processes. Recently, we have identified a pepducin (compound KRX-725-II) acting as an S1P3 receptor antagonist. Here, aiming to optimize the activity and selectivity profile of the described compound, we have synthesized a series of derivatives in which Tyr, in position 4, has been substituted with several natural aromatic and unnatural aromatic and non-aromatic amino acids. All the compounds were evaluated for their ability to inhibit vascular relaxation induced by KRX-725 (as S1P3 selective pepducin agonist) and KRX-722 (an S1P1-selective pepducin agonist). Those selective towards S1P3 (compounds V and VII) were also evaluated for their ability to inhibit skeletal muscle fibrosis. Finally, molecular dynamics simulations were performed to derive information on the preferred conformations of selective and unselective antagonists.

Oxidative Stress and ROS-Mediated Signaling in Leukemia: Novel Promising Perspectives to Eradicate Chemoresistant Cells in Myeloid Leukemia.

Myeloid leukemic cells are intrinsically under oxidative stress due to impaired reactive oxygen species (ROS) homeostasis, a common signature of several hematological malignancies. The present review focuses on the molecular mechanisms of aberrant ROS production in myeloid leukemia cells as well as on the redox-dependent signaling pathways involved in the leukemogenic process. Finally, the relevance of new chemotherapy options that specifically exert their pharmacological activity by altering the cellular redox imbalance will be discussed as an effective strategy to eradicate chemoresistant cells.

Discovery of a Novel Inhibitor of Human Purine Nucleoside Phosphorylase by a Simple Hydrophilic Interaction Liquid Chromatography Enzymatic Assay.

Human purine nucleoside phosphorylase (HsPNP) belongs to the purine salvage pathway of nucleic acids. Genetic deficiency of this enzyme triggers apoptosis of activated T-cells due to the accumulation of deoxyguanosine triphosphate (dGTP). Therefore, potential chemotherapeutic applications of human PNP inhibitors include the treatment of T-cell leukemia, autoimmune diseases and transplant tissue rejection. In this report, we present the discovery of novel HsPNP inhibitors by coupling experimental and computational tools. A simple, inexpensive, direct and non-radioactive enzymatic assay coupled to hydrophilic interaction liquid chromatography and UV detection (LC-UV using HILIC as elution mode) was developed for screening HsPNP inhibitors. Enzymatic activity was assessed by monitoring the phosphorolysis of inosine (Ino) to hypoxanthine (Hpx) by LC-UV. A small library of 6- and 8-substituted nucleosides was synthesized and screened. The inhibition potency of the most promising compound, 8-aminoinosine (4), was quantified through Ki and IC50 determinations. The effect of HsPNP inhibition was also evaluated in vitro through the study of cytotoxicity on human T-cell leukemia cells (CCRF-CEM). Docking studies were also carried out for the most potent compound, allowing further insights into the inhibitor interaction at the HsPNP active site. This study provides both new tools and a new lead for developing novel HsPNP inhibitors.

Inhibition of 37/67kDa Laminin-1 Receptor Restores APP Maturation and Reduces Amyloid-ß in Human Skin Fibroblasts from Familial Alzheimer's Disease.

Alzheimer's disease (AD) is a fatal neurodegenerative disorder caused by protein misfolding and aggregation, affecting brain function and causing dementia. Amyloid beta (Aß), a peptide deriving from amyloid precursor protein (APP) cleavage by-and γ-secretases, is considered a pathological hallmark of AD. Our previous study, together with several lines of evidence, identified a strict link between APP, Aß and 37/67kDa laminin receptor (LR), finding the possibility to regulate intracellular APP localization and maturation through modulation of the receptor. Here, we report that in fibroblasts from familial AD (fAD), APP was prevalently expressed as an immature isoform and accumulated preferentially in the transferrin-positive recycling compartment rather than in the Golgi apparatus. Moreover, besides the altered mitochondrial network exhibited by fAD patient cells, the levels of pAkt and pGSK3 were reduced in respect to healthy control fibroblasts and were accompanied by an increased amount of secreted Aß in conditioned medium from cell cultures. Interestingly, these features were reversed by inhibition of 37/67kDa LR by NSC47924 a small molecule that was able to rescue the "typical" APP localization in the Golgi apparatus, with consequences on the Aß level and mitochondrial network. Altogether, these findings suggest that 37/67kDa LR modulation may represent a useful tool to control APP trafficking and Aß levels with implications in Alzheimer's disease.