Publications by authors named "Giorgia Federico"

12 Publications

  • Page 1 of 1

The pyruvate kinase activator mitapivat reduces hemolysis and improves anemia in a β-thalassemia mouse model.

J Clin Invest 2021 Apr 6. Epub 2021 Apr 6.

Department of Medicine, University of Verona, Verona, Italy.

Anemia in β-thalassemia is related to ineffective erythropoiesis and reduced red cell survival. Excess free heme and accumulation of unpaired α-globin chains impose substantial oxidative stress on β-thalassemic erythroblasts and erythrocytes, impacting cell metabolism. We hypothesized that increased pyruvate kinase activity induced by mitapivat (AG-348) in the Hbbth3/+ mouse model for β-thalassemia would reduce chronic hemolysis and ineffective erythropoiesis through stimulation of red cell glycolytic metabolism. Oral mitapivat administration ameliorated ineffective erythropoiesis and anemia in Hbbth3/+ mice. Increased ATP, reduced reactive oxygen species production, and reduced markers of mitochondrial dysfunction associated with improved mitochondrial clearance suggested enhanced metabolism following mitapivat administration in β-thalassemia. The amelioration of responsiveness to erythropoietin resulted in reduced soluble erythroferrone, increased liver Hamp expression, and diminished liver iron overload. Mitapivat reduced duodenal Dmt1 expression potentially by activating the pyruvate kinase M2HIF2α axis, representing a mechanism additional to Hamp in controlling iron absorption and preventing β-thalassemia-related liver iron overload. In ex vivo studies on erythroid precursors from patients with β-thalassemia, mitapivat enhanced erythropoiesis, promoted erythroid maturation, and decreased apoptosis. Overall, pyruvate kinase activation as a treatment modality for β-thalassemia in preclinical model systems had multiple beneficial effects in the erythropoietic compartment and beyond, providing a strong scientific basis for further clinical trials.
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http://dx.doi.org/10.1172/JCI144206DOI Listing
April 2021

RET Gene Fusions in Malignancies of the Thyroid and Other Tissues.

Genes (Basel) 2020 04 15;11(4). Epub 2020 Apr 15.

Department of Molecular Medicine and Medical Biotechnology, University of Naples "Federico II", 80131 Naples, Italy.

Following the identification of the BCR-ABL1 (Breakpoint Cluster Region-ABelson murine Leukemia) fusion in chronic myelogenous leukemia, gene fusions generating chimeric oncoproteins have been recognized as common genomic structural variations in human malignancies. This is, in particular, a frequent mechanism in the oncogenic conversion of protein kinases. Gene fusion was the first mechanism identified for the oncogenic activation of the receptor tyrosine kinase RET (REarranged during Transfection), initially discovered in papillary thyroid carcinoma (PTC). More recently, the advent of highly sensitive massive parallel (next generation sequencing, NGS) sequencing of tumor DNA or cell-free (cfDNA) circulating tumor DNA, allowed for the detection of RET fusions in many other solid and hematopoietic malignancies. This review summarizes the role of RET fusions in the pathogenesis of human cancer.
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http://dx.doi.org/10.3390/genes11040424DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7230609PMC
April 2020

Bioisosteric Discovery of NPA101.3, a Second-Generation RET/VEGFR2 Inhibitor Optimized for Single-Agent Polypharmacology.

J Med Chem 2020 05 28;63(9):4506-4516. Epub 2020 Apr 28.

Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università di Napoli "Federico II", 80131 Napoli, Italy.

RET receptor tyrosine kinase is a driver oncogene in human cancer. We recently identified the clinical drug candidate Pz-1, which targets RET and VEGFR2. A key metabolite of Pz-1 is its less active demethylated pyrazole analogue. Using bioisosteric substitution methods, here, we report the identification of NPA101.3, lacking the structural liability for demethylation. NPA101.3 showed a selective inhibitory profile and an inhibitory concentration 50 (IC) of <0.003 μM for both RET and VEGFR2. NPA101.3 inhibited phosphorylation of all tested RET oncoproteins as well as VEGFR2 and proliferation of cells transformed by RET. Oral administration of NPA101.3 (10 mg/kg/day) completely prevented formation of tumors induced by RET/C634Y-transformed cells, while it weakened, but did not abrogate, formation of tumors induced by a control oncogene (HRAS/G12V). The balanced synchronous inhibition of both RET and VEGFR2, as well the resistance to demethylation, renders NPA101.3 a potential clinical candidate for RET-driven cancers.
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http://dx.doi.org/10.1021/acs.jmedchem.9b01336DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7901654PMC
May 2020

NCOA4-mediated ferritinophagy in macrophages is crucial to sustain erythropoiesis in mice.

Haematologica 2021 03 1;106(3):795-805. Epub 2021 Mar 1.

Department of Molecular Medicine and Medicine Biotechnology (DMMBM), University of Naples Federico II, Naples

Nuclear receptor coactivator 4 (NCOA4) promotes ferritin degradation and Ncoa4-ko mice in a C57BL/6 background show microcytosis and mild anemia, aggravated by iron deficiency. To understand tissue-specific contributions of NCOA4-mediated ferritinophagy we explored the effect of Ncoa4 genetic ablation in the iron-rich Sv129/J strain. Increased body iron content protects these mice from anemia and, in basal conditions, Sv129/J Ncoa4-ko mice show only microcytosis; nevertheless, when fed a low-iron diet they develop a more severe anemia compared to that of wild-type animals. Reciprocal bone marrow (BM) transplantation from wild-type donors into Ncoa4-ko and from Ncoa4-ko into wild-type mice revealed that microcytosis and susceptibility to iron deficiency anemia depend on BM-derived cells. Reconstitution of erythropoiesis with normalization of red blood count and hemoglobin concentration occurred at the same rate in transplanted animals independently of the genotype. Importantly, NCOA4 loss did not affect terminal erythropoiesis in iron deficiency, both in total and specific BM Ncoa4-ko animals compared to controls. On the contrary, upon a low iron diet, spleen from wild-type animals with Ncoa4-ko BM displayed marked iron retention compared to (wild-type BM) controls, indicating defective macrophage iron release in the former. Thus, erythropoietin administration failed to mobilize iron from stores in Ncoa4-ko animals. Furthermore, Ncoa4 inactivation in thalassemic mice did not worsen the hematologic phenotype. Overall our data reveal a major role for NCOA4-mediated ferritinophagy in macrophages to favor iron release for erythropoiesis, especially in iron deficiency.
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http://dx.doi.org/10.3324/haematol.2019.241232DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7928015PMC
March 2021

Iron and Ferritin Modulate MHC Class I Expression and NK Cell Recognition.

Front Immunol 2019 26;10:224. Epub 2019 Feb 26.

Tumor Immunology and Immunopathology Laboratory, Department of Experimental and Clinical Medicine, University Magna Graecia of Catanzaro, Catanzaro, Italy.

The ability of pathogens to sequester iron from their host cells and proteins affects their virulence. Moreover, iron is required for various innate host defense mechanisms as well as for acquired immune responses. Therefore, intracellular iron concentration may influence the interplay between pathogens and immune system. Here, we investigated whether changes in iron concentrations and intracellular ferritin heavy chain (FTH) abundance may modulate the expression of Major Histocompatibility Complex molecules (MHC), and susceptibility to Natural Killer (NK) cell cytotoxicity. FTH downregulation, either by shRNA transfection or iron chelation, led to MHC surface reduction in primary cancer cells and macrophages. On the contrary, mouse embryonic fibroblasts (MEFs) from NCOA4 null mice accumulated FTH for ferritinophagy impairment and displayed MHC class I cell surface overexpression. Low iron concentration, but not FTH, interfered with IFN-γ receptor signaling, preventing the increase of MHC-class I molecules on the membrane by obstructing STAT1 phosphorylation and nuclear translocation. Finally, iron depletion and FTH downregulation increased the target susceptibility of both primary cancer cells and macrophages to NK cell recognition. In conclusion, the reduction of iron and FTH may influence the expression of MHC class I molecules leading to NK cells activation.
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http://dx.doi.org/10.3389/fimmu.2019.00224DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6404638PMC
September 2020

Peroxiredoxin-2: A Novel Regulator of Iron Homeostasis in Ineffective Erythropoiesis.

Antioxid Redox Signal 2018 Jan 6;28(1):1-14. Epub 2017 Sep 6.

1 Department of Medicine, University of Verona-AOUI Verona , Verona, Italy .

Aims: Iron overload (IO) is a life-threatening complication of chronic hemolytic disorders such as β-thalassemia. IO results in severe cellular oxidative damage, leading to organ failure. Peroxiredoxin-2 (Prx2), a typical 2-cysteine-(Cys)-peroxiredoxin, is an important component of the cytoprotective system, but its response to IO is still to be fully defined.

Results: We studied the effects of IO on Prx2-knockout mice (Prx2). The absence of Prx2 enhanced toxicity due to IO on erythropoiesis. We found that IO failed to induce the typical hepcidin (Hamp) upregulation in Prx2 mice due to its failure to activate the signal transducer and activator of transcription-3 (STAT3) with intact Jak2 signaling. In Prx2 mice, the loss of Hamp response was also observed after administration of a single dose of oral iron. When lipopolysaccharide (LPS) was used to explore IL6-STAT3 activation in Prx2 mice, STAT3 activation and Hamp upregulation were once again defective. Treatment with PEP-fusion-recombinant-Prx2 (PEP Prx2) significantly increased STAT3 activation with upregulation of Hamp expression in both IO- and LPS-exposed Prx2 mice. We also confirmed the beneficial effects of PEP Prx2 on Hamp expression through STAT3 activation in β-thalassemic mice.

Innovation: We propose that Prx2 plays a key role in responding to cytotoxicity of IO, directly targeting STAT3-transcriptional factor in a Jak2-independent fashion and regulating Hamp in response to canonical stimuli.

Conclusion: Collectively, our data highlight a novel role of Prx2 in iron homeostasis. Prx2 is a key cytoprotector against IO that is induced either by iron supplementation or due to chronic hemolysis as in β-thalassemia. Prx2 is required to support STAT3 transcriptional activity and regulation of Hamp expression. Antioxid. Redox Signal. 28, 1-14.
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http://dx.doi.org/10.1089/ars.2017.7051DOI Listing
January 2018

The chemokine scavenging receptor D6/ACKR2 is a target of miR-146a in thyroid cancer.

Genes Cancer 2017 May;8(5-6):577-588

Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Federico II University, Naples, Italy.

We have previously shown that miR-146a, a NF-κB-regulated microRNA, is strongly expressed in human specimens and cell lines derived from anaplastic thyroid carcinomas (ATC) where it mediates some of the NF-κB pro-tumorigenic functions. By using a bioinformatic analysis, we identified the chemokine scavenger receptor D6/ ACKR2 as a target of miR146a in human ATC. We found that the expression of D6/ ACKR2 was up-regulated in miR-146a-null ATC cell lines and that the 3' UTR of D6/ ACKR2 mRNA was able to inhibit its expression in parental, but not in miR-146a-null ATC cells. Since human specimens from primary ATC showed a low expression of D6/ ACKR2 compared to normal thyroid tissues, we analyzed the effects of D6/ACKR2 over-expression in ATC cells. Different chemokines added to the conditioned medium of D6/ACKR2 over-expressing ATC cells partially failed to drive monocyte migration, and tumors derived from the injection of the same cells in nude mice showed a decreased number of infiltrating macrophages. Taken together, these results indicate that ATC cells down-regulate D6/ACKR2 expression through miR-146a activity to sustain leukocyte trafficking inside tumor microenvironment and shed light on a novel mechanism by which NF-κB indirectly inhibits the expression and the function of anti-tumorigenic gene in thyroid cancer.
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http://dx.doi.org/10.18632/genesandcancer.141DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5511891PMC
May 2017

Oncogene-induced senescence and its evasion in a mouse model of thyroid neoplasia.

Mol Cell Endocrinol 2018 01 23;460:24-35. Epub 2017 Jun 23.

Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Universita' Federico II c/o Istituto di Endocrinologia e Oncologia Sperimentale, CNR, Via S Pansini 5, 80131 Naples, Italy. Electronic address:

Here we describe a conditional doxycycline-dependent mouse model of RET/PTC3 (NCOA4-RET) oncogene-induced thyroid tumorigenesis. In these mice, after 10 days of doxycycline (dox) administration, RET/PTC3 expression induced mitogen activated protein kinase (MAPK) stimulation and a proliferative response which resulted in the formation of hyperplastic thyroid lesions. This was followed, after 2 months, by growth arrest accompanied by typical features of oncogene-induced senescence (OIS), including upregulation of p16INK4A and p21CIP, positivity at the Sudan black B, activation of the DNA damage response (DDR) markers γH2AX and pChk2 T68, and induction of p53 and p19ARF. After 5 months, about half of thyroid lesions escaped OIS and formed tumors that remained dependent on RET/PTC3 expression. This progression was accompanied by activation of AKT-FOXO1/3a pathway and increased serum TSH levels.
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http://dx.doi.org/10.1016/j.mce.2017.06.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5741508PMC
January 2018

NCOA4 Deficiency Impairs Systemic Iron Homeostasis.

Cell Rep 2016 Jan 14;14(3):411-421. Epub 2016 Jan 14.

Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università degli Studi di Napoli Federico II, and Istituto di Endocrinologia ed Oncologia Sperimentale del Consiglio Nazionale delle Ricerche, 80131 Napoli, Italy. Electronic address:

The cargo receptor NCOA4 mediates autophagic ferritin degradation. Here we show that NCOA4 deficiency in a knockout mouse model causes iron accumulation in the liver and spleen, increased levels of transferrin saturation, serum ferritin, and liver hepcidin, and decreased levels of duodenal ferroportin. Despite signs of iron overload, NCOA4-null mice had mild microcytic hypochromic anemia. Under an iron-deprived diet (2-3 mg/kg), mice failed to release iron from ferritin storage and developed severe microcytic hypochromic anemia and ineffective erythropoiesis associated with increased erythropoietin levels. When fed an iron-enriched diet (2 g/kg), mice died prematurely and showed signs of liver damage. Ferritin accumulated in primary embryonic fibroblasts from NCOA4-null mice consequent to impaired autophagic targeting. Adoptive expression of the NCOA4 COOH terminus (aa 239-614) restored this function. In conclusion, NCOA4 prevents iron accumulation and ensures efficient erythropoiesis, playing a central role in balancing iron levels in vivo.
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http://dx.doi.org/10.1016/j.celrep.2015.12.065DOI Listing
January 2016

Fragment-Based Discovery of a Dual pan-RET/VEGFR2 Kinase Inhibitor Optimized for Single-Agent Polypharmacology.

Angew Chem Int Ed Engl 2015 Jul 30;54(30):8717-21. Epub 2015 Jun 30.

Medicinal Chemistry Division, Pharmacology and Toxicology, College of Pharmacy, The University of Arizona, 1703 E. Mabel, Tucson, AZ 85721 (USA).

Oncogenic conversion of the RET (rearranged during transfection) tyrosine kinase is associated with several cancers. A fragment-based chemical screen led to the identification of a novel RET inhibitor, Pz-1. Modeling and kinetic analysis identified Pz-1 as a type II tyrosine kinase inhibitor that is able to bind the "DFG-out" conformation of the kinase. Importantly, from a single-agent polypharmacology standpoint, Pz-1 was shown to be active on VEGFR2, which can block the blood supply required for RET-stimulated growth. In cell-based assays, 1.0 nM of Pz-1 strongly inhibited phosphorylation of all tested RET oncoproteins. At 1.0 mg kg(-1)  day(-1) per os, Pz-1 abrogated the formation of tumors induced by RET-mutant fibroblasts and blocked the phosphorylation of both RET and VEGFR2 in tumor tissue. Pz-1 featured no detectable toxicity at concentrations of up to 100.0 mg kg(-1), which indicates a large therapeutic window. This study validates the effectiveness and usefulness of a medicinal chemistry/polypharmacology approach to obtain an inhibitor capable of targeting multiple oncogenic pathways.
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http://dx.doi.org/10.1002/anie.201501104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4535927PMC
July 2015

Identification of Novel Small Molecule Inhibitors of Oncogenic RET Kinase.

PLoS One 2015 5;10(6):e0128364. Epub 2015 Jun 5.

Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università di Napoli "Federico II", Naples, Italy; Istituto di Endocrinologia ed Oncologia Sperimentale del CNR, Naples, Italy.

Oncogenic mutation of the RET receptor tyrosine kinase is observed in several human malignancies. Here, we describe three novel type II RET tyrosine kinase inhibitors (TKI), ALW-II-41-27, XMD15-44 and HG-6-63-01, that inhibit the cellular activity of oncogenic RET mutants at two digit nanomolar concentration. These three compounds shared a 3-trifluoromethyl-4-methylpiperazinephenyl pharmacophore that stabilizes the 'DFG-out' inactive conformation of RET activation loop. They blocked RET-mediated signaling and proliferation with an IC50 in the nM range in fibroblasts transformed by the RET/C634R and RET/M918T oncogenes. They also inhibited autophosphorylation of several additional oncogenic RET-derived point mutants and chimeric oncogenes. At a concentration of 10 nM, ALW-II-41-27, XMD15-44 and HG-6-63-01 inhibited RET kinase and signaling in human thyroid cancer cell lines carrying oncogenic RET alleles; they also inhibited proliferation of cancer, but not non-tumoral Nthy-ori-3-1, thyroid cells, with an IC50 in the nM range. The three compounds were capable of inhibiting the 'gatekeeper' V804M mutant which confers substantial resistance to established RET inhibitors. In conclusion, we have identified a type II TKI scaffold, shared by ALW-II-41-27, XMD15-44 and HG-6-63-01, that may be used as novel lead for the development of novel agents for the treatment of cancers harboring oncogenic activation of RET.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0128364PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4457528PMC
March 2016