Publications by authors named "Francesca Carlomagno"

59 Publications

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

Discovery of pyrazolo-thieno[3,2-d]pyrimidinylamino-phenyl acetamides as type-II pan-tropomyosin receptor kinase (TRK) inhibitors: Design, synthesis, and biological evaluation.

Eur J Med Chem 2021 Apr 9;216:113265. Epub 2021 Feb 9.

Department of Pharmaceutical Sciences, College of Pharmacy, University of Arkansas for Medical Sciences, Little Rock, AR, 72205, USA. Electronic address:

Tropomyosin receptor kinase (TRK) represents an attractive oncology target for cancer therapy related to its critical role in cancer formation and progression. NTRK fusions are found to occur in 3.3% of lung cancers, 2.2% of colorectal cancers, 16.7% of thyroid cancers, 2.5% of glioblastomas, and 7.1% of pediatric gliomas. In this paper, we described the discovery of the type-II pan-TRK inhibitor 4c through the structure-based drug design strategy from the original hits 1b and 2b. Compound 4c exhibited excellent in vitro TRKA, TRKB, and TRKC kinase inhibitory activity and anti-proliferative activity against human colorectal carcinoma derived cell line KM12. In the NCI-60 human cancer cell lines screen, compound 4g demonstrated nearly 80% of growth inhibition for KM12, while only minimal inhibitory activity was observed for the remaining 59 cancer cell lines. Western blot analysis demonstrated that 4c and its urea cousin 4k suppressed the TPM3-TRKA autophosphorylation at the concentrations of 100 nM and 10 nM, respectively. The work presented that 2-(4-(thieno[3,2-d]pyrimidin-4-ylamino)phenyl)acetamides could serve as a novel scaffold for the discovery and development of type-II pan-TRK inhibitors for the treatment of TRK driven cancers.
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http://dx.doi.org/10.1016/j.ejmech.2021.113265DOI 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

Association between DNA methylation profile and malignancy in follicular-patterned thyroid neoplasms.

Endocr Relat Cancer 2019 04 1;26(4):451-462. Epub 2019 Feb 1.

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

Molecular differentiation between benign (follicular thyroid adenoma, FTA) and malignant (follicular thyroid carcinoma, FTC) thyroid neoplasms is challenging. Here, we explored the genome-wide DNA methylation profile of FTA (n.10) and FTC (n.11) compared to normal thyroid (NT) (n.7) tissues. FTC featured 3,564 differentially-methylated CpGs (DMCpG), most (84%) of them hypermethylated, with respect to normal controls. At the principal component analysis (PCA), the methylation profile of FTA occupied an intermediate position between FTC and normal tissue. A large fraction (n. 2,385) of FTC-associated DMCpG were related (intragenic or within 1500 bp from the transcription start site) to annotated genes (n. 1,786). FTC-hypermethylated genes were enriched for targets of the Polycomb transcriptional repressor complex and the specific histone H3 marks (H3K4me2/me3-H3K27me3) found in chromatin domains known as "bivalent". Transcriptome profiling by RNAseq showed that 7.9% of the DMCpGs-associated genes were differentially expressed in FTC compared to NT, suggesting that altered DNA methylation may contribute to their altered expression. Overall, this study suggests that perturbed DNA methylation, in particular hypermethylation, is a component of the molecular mechanisms leading to the formation of FTC and that DNA methylation profiling may help differentiating FTCs from their benign counterpart.
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http://dx.doi.org/10.1530/ERC-18-0308DOI Listing
April 2019

Insights into Current Tropomyosin Receptor Kinase (TRK) Inhibitors: Development and Clinical Application.

J Med Chem 2019 02 19;62(4):1731-1760. Epub 2018 Sep 19.

Department of Pharmaceutical Sciences, College of Pharmacy , University of Arkansas for Medical Sciences , Little Rock , Arkansas 72205 , United States.

The use of kinase-directed precision medicine has been heavily pursued since the discovery and development of imatinib. Annually, it is estimated that around ∼20 000 new cases of tropomyosin receptor kinase (TRK) cancers are diagnosed, with the majority of cases exhibiting a TRK genomic rearrangement. In this Perspective, we discuss current development and clinical applications for TRK precision medicine by providing the following: (1) the biological background and significance of the TRK kinase family, (2) a compilation of known TRK inhibitors and analysis of their cocrystal structures, (3) an overview of TRK clinical trials, and (4) future perspectives for drug discovery and development of TRK inhibitors.
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http://dx.doi.org/10.1021/acs.jmedchem.8b01092DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7875308PMC
February 2019

The molecular basis for RET tyrosine-kinase inhibitors in thyroid cancer.

Best Pract Res Clin Endocrinol Metab 2017 06 10;31(3):307-318. Epub 2017 May 10.

Istituto di Endocrinologia e Oncologia Sperimentale, CNR, Via S Pansini 5, 80131 Naples, Italy; Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università Federico II, Via S Pansini 5, 80131 Naples, Italy. Electronic address:

RET receptor tyrosine kinase acts as a mutated oncogenic driver in several human malignancies and it is over-expressed in other cancers. Small molecule compounds with RET tyrosine kinase inhibitory activity are being investigated for the targeted treatment of these malignancies. Multi-targeted compounds with RET inhibitory concentration in the nanomolar range have entered clinical practice. This review summarizes mechanisms of RET oncogenic activity and properties of new compounds that, at the preclinical stage, have demonstrated promising anti-RET activity.
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http://dx.doi.org/10.1016/j.beem.2017.04.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5624797PMC
June 2017

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

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

Identification of two novel RET kinase inhibitors through MCR-based drug discovery: design, synthesis and evaluation.

Eur J Med Chem 2014 Oct 8;86:714-23. Epub 2014 Sep 8.

College of Pharmacy, Department of Pharmacology and Toxicology, The University of Arizona, Tucson, AZ 85721, USA; The University of Arizona Cancer Center, 1515 N Campbell Ave, Tucson, AZ 85724, USA. Electronic address:

From an MCR fragment library, two novel chemical series have been developed as inhibitors of RET, which is a kinase involved in the pathology of medullary thyroid cancer (MTC). Structure activity relationship studies (SAR) identified two sub-micromolar tractable leads, 6g and 13g. 6g was confirmed to be a Type-II RET inhibitor. 13g and 6g inhibited RET in cells transformed by RET/C634. A RET DFG-out homology model was established and utilized to predict Type-II inhibitor binding modes.
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http://dx.doi.org/10.1016/j.ejmech.2014.09.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4666024PMC
October 2014

NCOA4 transcriptional coactivator inhibits activation of DNA replication origins.

Mol Cell 2014 Jul 5;55(1):123-37. Epub 2014 Jun 5.

Istituto di Endocrinologia ed Oncologia Sperimentale del CNR/Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università di Napoli "Federico II," 80131 Naples, Italy. Electronic address:

NCOA4 is a transcriptional coactivator of nuclear hormone receptors that undergoes gene rearrangement in human cancer. By combining studies in Xenopus laevis egg extracts and mouse embryonic fibroblasts (MEFs), we show here that NCOA4 is a minichromosome maintenance 7 (MCM7)-interacting protein that is able to control DNA replication. Depletion-reconstitution experiments in Xenopus laevis egg extracts indicate that NCOA4 acts as an inhibitor of DNA replication origin activation by regulating CMG (CDC45/MCM2-7/GINS) helicase. NCOA4(-/-) MEFs display unscheduled origin activation and reduced interorigin distance; this results in replication stress, as shown by the presence of fork stalling, reduction of fork speed, and premature senescence. Together, our findings indicate that NCOA4 acts as a regulator of DNA replication origins that helps prevent inappropriate DNA synthesis and replication stress.
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http://dx.doi.org/10.1016/j.molcel.2014.04.031DOI Listing
July 2014

Growth factor transduction pathways: paradigm of anti-neoplastic targeted therapy.

J Mol Med (Berl) 2014 Jul 8;92(7):723-33. Epub 2014 Jun 8.

Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università degli Studi di Napoli Federico II, via Pansini 5, 80131, Naples, Italy,

Molecularly targeted cancer treatment has become an achievable goal thanks to systematic analysis of cancer genome as well as development of highly selective tumor targeted drugs. In many human cancers, deregulation of the RTK/RAS/MAPK pathway is the driving force of the disease. Indeed, cancer cells become addicted to such signaling, rendering them susceptible to drugs that can intercept growth factor signaling cascade at different levels. Discovery of mutations or aberrant expression of components of this cascade in radio- and chemotherapy refractory human melanoma acted as an enormous stimulus for scientist to try to identify and clinically test new therapeutic approaches blocking the RTK/RAS/MAPK cascade. These efforts not only resulted in the identification of new drugs for melanoma treatment but also in a better understanding of molecular basis of primary and secondary resistance to targeted therapies.
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http://dx.doi.org/10.1007/s00109-014-1177-7DOI Listing
July 2014

Functional characterization of a novel FGFR1OP-RET rearrangement in hematopoietic malignancies.

Mol Oncol 2014 Mar 19;8(2):221-31. Epub 2013 Nov 19.

Department of Experimental Oncology, IEO, European Institute of Oncology, Milan, Italy; Department of Biosciences, University of Milan, Milan, Italy. Electronic address:

The RET (REarranged during Transfection) receptor tyrosine kinase is targeted by oncogenic rearrangements in thyroid and lung adenocarcinoma. Recently, a RET (exon 12) rearrangement with FGFR1OP [fibroblast growth factor receptor 1 (FGFR1) oncogene partner] (exon 12) was identified in one chronic myelomonocytic leukemia (CMML) patient. We report the molecular cloning and functional characterization of a novel FGFR1OP (exon 11)-RET (exon 11) gene fusion event (named FGFR1OP-RET), mediated by a reciprocal translocation t(6; 10)(q27; q11), in a patient affected by primary myelofibrosis (PMF) with secondary acute myeloid leukemia (AML). The FGFR1OP-RET fusion protein displayed constitutive tyrosine kinase and transforming activity in NIH3T3 fibroblasts, and induced IL3-independent growth and activation of PI3K/STAT signaling in hematopoietic Ba/F3 cells. FGFR1OP-RET supported cytokine-independent growth, protection from stress and enhanced self-renewal of primary murine hematopoietic progenitor and stem cells in vitro. In vivo, FGFR1OP-RET caused a spectrum of disease phenotypes, with >50% of mice showing a fatal myeloproliferative disorder (MPD). Other phenotypes were leukemia transplantable in secondary recipients, dramatic expansion of the mast cell lineage, and reduction of repopulating activity upon lethal irradiation. In conclusion, FGFR1OP-RET chimeric oncogenes are endowed with leukemogenic potential and associated to myeloid neoplasms (CMML and PMF/AML).
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http://dx.doi.org/10.1016/j.molonc.2013.11.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5528547PMC
March 2014

Central role of RET in thyroid cancer.

Cold Spring Harb Perspect Biol 2013 Dec 1;5(12):a009233. Epub 2013 Dec 1.

Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Universita' degli Studi di Napoli Federico II, 80131 Napoli, Italy.

RET (rearranged during transfection) is a receptor tyrosine kinase involved in the development of neural crest derived cell lineages, kidney, and male germ cells. Different human cancers, including papillary and medullary thyroid carcinomas, lung adenocarcinomas, and myeloproliferative disorders display gain-of-function mutations in RET. Accordingly, RET protein has become a promising molecular target for cancer treatment.
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http://dx.doi.org/10.1101/cshperspect.a009233DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3839608PMC
December 2013

Oncogenic rearrangements driving ionizing radiation-associated human cancer.

J Clin Invest 2013 Nov 25;123(11):4566-8. Epub 2013 Oct 25.

The Chernobyl nuclear disaster has caused a remarkable increase in radiation-induced papillary thyroid carcinoma in children and young adults. In this issue of the JCI, Ricarte-Filho and colleagues demonstrate that chromosomal rearrangements are the oncogenic "drivers" in most post-Chernobyl carcinomas and that they often lead to unscheduled activation of the MAPK signaling pathway. These findings represent a major step forward in our understanding of radiation-induced carcinogenesis and suggest various hypotheses about the mechanisms underlying the formation and selection of gene rearrangements during cancer cell evolution.
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http://dx.doi.org/10.1172/JCI72725DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3809810PMC
November 2013

Attacking cancer with molecularly targeted agents.

Curr Opin Pharmacol 2013 Aug 16;13(4):483-5. Epub 2013 Jul 16.

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http://dx.doi.org/10.1016/j.coph.2013.06.011DOI Listing
August 2013

Molecular mechanism of 17-allylamino-17-demethoxygeldanamycin (17-AAG)-induced AXL receptor tyrosine kinase degradation.

J Biol Chem 2013 Jun 29;288(24):17481-94. Epub 2013 Apr 29.

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

The receptor tyrosine kinase AXL is overexpressed in many cancer types including thyroid carcinomas and has well established roles in tumor formation and progression. Proper folding, maturation, and activity of several oncogenic receptor tyrosine kinases require HSP90 chaperoning. HSP90 inhibition by the antibiotic geldanamycin or its derivative 17-allylamino-17-demethoxygeldanamycin (17-AAG) causes destabilization of its client proteins. Here we show that AXL is a novel client protein of HSP90. 17-AAG induced a time- and dose-dependent down-regulation of endogenous or ectopically expressed AXL protein, thereby inhibiting AXL-mediated signaling and biological activity. 17-AAG-induced AXL down-regulation specifically affected fully glycosylated mature receptor present on cell membrane. By using biotin and [(35)S]methionine labeling, we showed that 17-AAG caused depletion of membrane-localized AXL by mediating its degradation in the intracellular compartment, thus restricting its exposure on the cell surface. 17-AAG induced AXL polyubiquitination and subsequent proteasomal degradation; under basal conditions, AXL co-immunoprecipitated with HSP90. Upon 17-AAG treatment, AXL associated with the co-chaperone HSP70 and the ubiquitin E3 ligase carboxyl terminus of HSC70-interacting protein (CHIP). Overexpression of CHIP, but not of the inactive mutant CHIP K30A, induced accumulation of AXL polyubiquitinated species upon 17-AAG treatment. The sensitivity of AXL to 17-AAG required its intracellular domain because an AXL intracellular domain-deleted mutant was insensitive to the compound. Active AXL and kinase-dead AXL were similarly sensitive to 17-AAG, implying that 17-AAG sensitivity does not require receptor phosphorylation. Overall our data elucidate the molecular basis of AXL down-regulation by HSP90 inhibitors and suggest that HSP90 inhibition in anticancer therapy can exert its effect through inhibition of multiple kinases including AXL.
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http://dx.doi.org/10.1074/jbc.M112.439422DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3682548PMC
June 2013

Ponatinib (AP24534) is a novel potent inhibitor of oncogenic RET mutants associated with thyroid cancer.

J Clin Endocrinol Metab 2013 May 22;98(5):E811-9. Epub 2013 Mar 22.

Istituto di Endocrinologia ed Oncologia Sperimentale del Consiglio Nazionale delle Ricerche, Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università di Napoli Federico II, 80131 Naples, Italy.

Context: The RET tyrosine kinase encoding gene acts as a dominantly transforming oncogene in thyroid carcinoma and other malignancies. Ponatinib (AP24534) is an oral ATP-competitive tyrosine kinase inhibitor that is in advanced clinical experimentation in leukemia.

Objective: We tested whether ponatinib inhibited RET kinase and oncogenic activity.

Methods: Ponatinib activity was studied by an in vitro RET immunocomplex kinase assay and immunoblotting. The effects of ponatinib on proliferation of human TT, MZ-CRC-1, and TPC-1 thyroid carcinoma cells, which harbor endogenous oncogenic RET alleles, and of NIH3T3 fibroblasts transfected with oncogenic RET mutants were determined. Ponatinib activity on TT cell xenografted tumors in athymic mice was measured.

Results: Ponatinib inhibited immunopurified RET kinase at the IC₅₀ of 25.8 nM (95% confidence interval [CI] = 23.15-28.77 nM). It also inhibited (IC₅₀ = 33.9 nM; 95% CI = 26.41-43.58 nM) kinase activity of RET/V804M, a RET mutant displaying resistance to other tyrosine kinase inhibitor. Ponatinib blunted phosphorylation of point-mutant and rearranged RET-derived oncoproteins and inhibited proliferation of RET-transformed fibroblasts and RET mutant thyroid carcinoma cells. Finally, after 3 weeks of treatment with ponatinib (30 mg/kg/d), the volume of TT cell (medullary thyroid carcinoma) xenografts was reduced from 133 mm³ to an unmeasurable size (difference = 133 mm³, 95% CI = -83 to 349 mm³) (P < .001). Ponatinib-treated TT cell tumors displayed a reduction in the mitotic index, RET phosphorylation, and signaling.

Conclusions: Ponatinib is a potent inhibitor of RET kinase and has promising preclinical activity in models of RET-driven medullary thyroid carcinoma.
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http://dx.doi.org/10.1210/jc.2012-2672DOI Listing
May 2013

Structure prediction and validation of the ERK8 kinase domain.

PLoS One 2013 11;8(1):e52011. Epub 2013 Jan 11.

Istituto Toscano Tumori-Core Research Laboratory, Signal Transduction Unit, AOU Senese, Siena, Italy.

Extracellular signal-regulated kinase 8 (ERK8) has been already implicated in cell transformation and in the protection of genomic integrity and, therefore, proposed as a novel potential therapeutic target for cancer. In the absence of a crystal structure, we developed a three-dimensional model for its kinase domain. To validate our model we applied a structure-based virtual screening protocol consisting of pharmacophore screening and molecular docking. Experimental characterization of the hit compounds confirmed that a high percentage of the identified scaffolds was able to inhibit ERK8. We also confirmed an ATP competitive mechanism of action for the two best-performing molecules. Ultimately, we identified an ERK8 drug-resistant "gatekeeper" mutant that corroborated the predicted molecular binding mode, confirming the reliability of the generated structure. We expect that our model will be a valuable tool for the development of specific ERK8 kinase inhibitors.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0052011PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3543423PMC
July 2013

Thyroid Cancer: Role of RET and Beyond.

Eur Thyroid J 2012 Apr 28;1(1):15-23. Epub 2012 Mar 28.

Dipartimento di Biologia e Patologia Cellulare e Molecolare L. Califano, Università degli Studi di Napoli Federico II, Napoli, Italia.

Specific thyroid cancer histotypes, such as papillary and medullary thyroid carcinoma, display genetic rearrangements or point mutations of the RET gene, resulting in its oncogenic conversion. The molecular mechanisms mediating RET rearrangement with other genes and the role of partner genes in tumorigenesis have been described. In addition, the RET protein has become a molecular target for medullary thyroid carcinoma treatment.
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http://dx.doi.org/10.1159/000336975DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3821455PMC
April 2012

Thyroid cancer in 2010: a roadmap for targeted therapies.

Nat Rev Endocrinol 2011 Feb;7(2):65-7

Dipartimento di Biologia e Patologia Cellulare e Molecolare L Califano, Universita' degli studi di Napoli, Via Pansini 5, 80131 Naples, Italy.

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http://dx.doi.org/10.1038/nrendo.2010.232DOI Listing
February 2011

The tyrosine kinase inhibitor ZD6474 blocks proliferation of RET mutant medullary thyroid carcinoma cells.

Endocr Relat Cancer 2011 Feb 30;18(1):1-11. Epub 2010 Nov 30.

Dipartimento di Biologia e Patologia Cellulare e Molecolare, Università Federico II, Napoli, Italy.

Oncogenic conversion of the RET tyrosine kinase is a frequent feature of medullary thyroid carcinoma (MTC). ZD6474 (vandetanib) is an ATP-competitive inhibitor of RET, epidermal growth factor receptor (EGFR), and vascular endothelial growth factor receptors kinases. In this study, we have studied ZD6474 mechanism of action in TT and MZ-CRC-1 human MTC cell lines, carrying cysteine 634 to tryptophan (C634W) and methionine 918 to threonine (M918T) RET mutation respectively. ZD6474 blunted MTC cell proliferation and RET, Shc and p44/p42 mitogen-activated protein kinase (MAPK) phosphorylation. Single receptor knockdown by RNA interference showed that MTC cells depended on RET for proliferation. Adoptive expression of the ZD6474-resistant V804M RET mutant rescued proliferation of TT cells under ZD6474 treatment, showing that RET is a key ZD6474 target in these MTC cells. Upon RET inhibition, adoptive stimulation of EGFR partially rescued TT cell proliferation, MAPK signaling, and expression of cell-cycle-related genes. This suggests that simultaneous inhibition of RET and EGFR by ZD6474 may overcome the risk of MTC cells to escape from RET blockade through compensatory over-activation of EGFR.
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http://dx.doi.org/10.1677/ERC-09-0292DOI Listing
February 2011

RET is a heat shock protein 90 (HSP90) client protein and is knocked down upon HSP90 pharmacological block.

J Clin Endocrinol Metab 2010 Jul 5;95(7):3552-7. Epub 2010 May 5.

Istituto di Endocrinologia ed Oncologia Sperimentale del Consiglio Nazionale delle Ricerche, Dipartimento di Biologia e Patologia Cellulare e Molecolare, Universitá Federico II, 80131 Naples, Italy.

Context: Mutations of the RET receptor tyrosine kinase are associated to multiple endocrine neoplasia type 2 (MEN2) and sporadic medullary thyroid carcinoma (MTC). The heat shock protein (HSP) 90 chaperone is required for folding and stability of several kinases. HSP90 is specifically inhibited by 17-allyl-amino-17-demethoxygeldanamycin (17-AAG).

Objective: Our aim was to investigate whether RET protein half-life depends on HSP90 and to dissect the molecular pathway responsible for the degradation of RET upon HSP90 inhibition by 17-AAG.

Design: 17-AAG effects were studied in RAT1 fibroblasts exogenously expressing MEN2-associated RET mutants and human MTC-derived cell lines.

Results: 17-AAG induced a 26S proteasome-dependent degradation of wild-type RET and MEN2-associated RET mutants. The compound hampered HSP90/RET interaction and stabilized RET binding to HSP70, leading to the recruitment of the HSP70-associated E3 ligase C-terminus of Hsc70-interacting protein. In turn, C-terminus of Hsc70-interacting protein polyubiquitinated RET, promoting its proteasomal degradation. 17-AAG blocked RET downstream effectors and RET-dependent transcriptional activation of gene promoters. In human MTC cells carrying oncogenic RET mutants, HSP90 inhibition induced receptor degradation and signaling hindrance leading to cell cycle arrest.

Conclusion: RET and MEN2-associated RET mutants rely on HSP90 for protein stability, and HSP90 blockade by 17-AAG promotes RET degradation.
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http://dx.doi.org/10.1210/jc.2009-2315DOI Listing
July 2010