miR-1202 acts as anti-oncomiR in myeloid leukaemia by down-modulating GATA-1S expression.

Transient abnormal myelopoiesis (TAM) is a Down syndrome-related pre-leukaemic condition characterized by somatic mutations in the haematopoietic transcription factor GATA-1 that result in exclusive production of its shorter isoform (GATA-1S). Given the common hallmark of altered miRNA expression profiles in haematological malignancies and the pro-leukaemic role of GATA-1S, we aimed to search for miRNAs potentially able to modulate the expression of GATA-1 isoforms. Starting from an in silico prediction of miRNA binding sites in the GATA-1 transcript, miR-1202 came into our sight as potential regulator of GATA-1 expression. Expression studies in K562 cells revealed that miR-1202 directly targets GATA-1, negatively regulates its expression, impairs GATA-1S production, reduces cell proliferation, and increases apoptosis sensitivity. Furthermore, data from TAM and myeloid leukaemia patients provided substantial support to our study by showing that miR-1202 down-modulation is accompanied by increased GATA-1 levels, with more marked effects on GATA-1S. These findings indicate that miR-1202 acts as an anti-oncomiR in myeloid cells and may impact leukaemogenesis at least in part by down-modulating GATA-1S levels.

Identification and Functional Analysis of Known and New Mutations in the Transcription Factor KLF1 Linked with ß-Thalassemia-like Phenotypes.

The erythroid transcriptional factor Krüppel-like factor 1 (KLF1) is a master regulator of erythropoiesis. Mutations that cause KLF1 haploinsufficiency have been linked to increased fetal hemoglobin (HbF) and hemoglobin A2 (HbA2) levels with ameliorative effects on the severity of ß-thalassemia. With the aim of determining if KLF1 gene variations might play a role in the modulation of ß-thalassemia, in this study we screened 17 subjects showing a ß-thalassemia-like phenotype with a slight or marked increase in HbA2 and HbF levels. Overall, seven KLF1 gene variants were identified, of which two were novel. Functional studies were performed in K562 cells to clarify the pathogenic significance of these mutations. Our study confirmed the ameliorative effect on the thalassemia phenotype for some of these variants but also raised the notion that certain mutations may have deteriorating effects by increasing KLF1 expression levels or enhancing its transcriptional activity. Our results indicate that functional studies are required to evaluate the possible effects of KLF1 mutations, particularly in the case of the co-existence of two or more mutations that could differently contribute to KLF1 expression or transcriptional activity and consequently to the thalassemia phenotype.

Over-Expressed GATA-1S, the Short Isoform of the Hematopoietic Transcriptional Factor GATA-1, Inhibits Ferroptosis in K562 Myeloid Leukemia Cells by Preventing Lipid Peroxidation.

Ferroptosis is a recently recognized form of regulated cell death involving lipid peroxidation. Glutathione peroxidase 4 (GPX4) plays a central role in the regulation of ferroptosis through the suppression of lipid peroxidation generation. Connections have been reported between ferroptosis, lipid metabolism, cancer onset, and drug resistance. Recently, interest has grown in ferroptosis induction as a potential strategy to overcome drug resistance in hematological malignancies. GATA-1 is a key transcriptional factor controlling hematopoiesis-related gene expression. Two GATA-1 isoforms, the full-length protein (GATA-1FL) and a shorter isoform (GATA-1S), are described. A balanced GATA-1FL/GATA-1S ratio helps to control hematopoiesis, with GATA-1S overexpression being associated with hematological malignancies by promoting proliferation and survival pathways in hematopoietic precursors. Recently, optical techniques allowed us to highlight different lipid profiles associated with the expression of GATA-1 isoforms, thus raising the hypothesis that ferroptosis-regulated processes could be involved. Lipidomic and functional analysis were conducted to elucidate these mechanisms. Studies on lipid peroxidation production, cell viability, cell death, and gene expression were used to evaluate the impact of GPX4 inhibition. Here, we provide the first evidence that over-expressed GATA-1S prevents K562 myeloid leukemia cells from lipid peroxidation-induced ferroptosis. Targeting ferroptosis is a promising strategy to overcome chemoresistance. Therefore, our results could provide novel potential therapeutic approaches and targets to overcome drug resistance in hematological malignancies.

Biological relevance of ZNF224 expression in chronic lymphocytic leukemia and its implication IN NF-kB pathway regulation.

Chronic lymphocytic leukemia (CLL) is a heterogeneous disease, whose presentation and clinical course are highly variable. Identification of novel prognostic factors may contribute to improving the CLL classification and providing indications for treatment options. The zinc finger protein ZNF224 plays a key role in cell transformation, through the control of apoptotic and survival pathways. In this study, we evaluated the potential application of ZNF224 as a novel marker of CLL progression and therapy responsiveness. To this aim, we analyzed ZNF224 expression levels in B lymphocytes from CLL patients at different stages of the disease and in patients showing different treatment outcomes. The expression of ZNF224 was significantly increased in disease progression and dramatically decreased in patients in complete remission after chemotherapy. Gene expression correlation analysis performed on datasets of CLL patients revealed that ZNF224 expression was well correlated with that of some prognostic and predictive markers. Moreover, bioinformatic analysis coupled ZNF224 to NF-κB pathway, and experimental data demonstrated that RNA interference of ZNF224 reduced the activity of the NF-κB survival pathway in CLL cells. Consistently with a pro-survival role, ZNF224 knockdown raised spontaneous and drug-induced apoptosis and inhibited the proliferation of peripheral blood mononuclear cells from CLL patients. Our findings provide evidence for the involvement of ZNF224 in the survival of CLL cells via NF-κB pathway modulation, and also suggest ZNF224 as a prognostic and predictive molecular marker of CLL disease.

Exploring a peptide nucleic acid-based antisense approach for CD5 targeting in chronic lymphocytic leukemia.

We herein report an innovative antisense approach based on Peptide Nucleic Acids (PNAs) to down-modulate CD5 expression levels in chronic lymphocytic leukemia (CLL). Using bioinformatics tools, we selected a 12-mer tract of the CD5 mRNA as the molecular target and synthesized the complementary and control PNA strands bearing a serine phosphate dipeptide tail to enhance their water solubility and bioavailability. The specific recognition of the 12-mer DNA strand, corresponding to the target mRNA sequence by the complementary PNA strand, was confirmed by non-denaturing polyacrylamide gel electrophoresis, thermal difference spectroscopy, circular dichroism (CD), and CD melting studies. Cytofluorimetric assays and real-time PCR analysis demonstrated the downregulation of CD5 expression due to incubation with the anti-CD5 PNA at RNA and protein levels in Jurkat cell line and peripheral blood mononuclear cells from B-CLL patients. Interestingly, we also observed that transfection with the anti-CD5 PNA increases apoptotic response induced by fludarabine in B-CLL cells. The herein reported results suggest that PNAs could represent a potential candidate for the development of antisense therapeutic agents in CLL.

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.

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.