RESUMO
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.
Assuntos
Eritropoese , Coativadores de Receptor Nuclear , Animais , Ferritinas , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Coativadores de Receptor Nuclear/genética , Coativadores de Receptor Nuclear/metabolismoRESUMO
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 typeâ 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, 1.0â nM of Pz-1 strongly inhibited phosphorylation of all tested RET oncoproteins. At 1.0â 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 100.0â 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.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-ret/antagonistas & inibidores , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Desenho de Fármacos , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Camundongos Nus , Modelos Moleculares , Células NIH 3T3 , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Fosforilação/efeitos dos fármacos , Polifarmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-ret/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Iron is essential for deoxyribonucleotides production and for enzymes containing an Fe-S cluster involved in DNA replication and repair. How iron bioavailability and DNA metabolism are coordinated remains poorly understood. NCOA4 protein mediates autophagic degradation of ferritin to maintain iron homeostasis and inhibits DNA replication origin activation via hindrance of the MCM2-7 DNA helicase. Here, we show that iron deficiency inhibits DNA replication, parallel to nuclear NCOA4 stabilization. In iron-depleted cells, NCOA4 knockdown leads to unscheduled DNA synthesis, with replication stress, genome instability, and cell death. In mice, NCOA4 genetic inactivation causes defective intestinal regeneration upon dextran sulfate sodium-mediated injury, with DNA damage, defective cell proliferation, and cell death; in intestinal organoids, this is fostered by iron depletion. In summary, we describe a NCOA4-dependent mechanism that coordinates iron bioavailability and DNA replication. This function prevents replication stress, maintains genome integrity, and sustains high rates of cell proliferation during tissue regeneration.
Assuntos
Ferro , Coativadores de Receptor Nuclear , Animais , Disponibilidade Biológica , DNA/metabolismo , Replicação do DNA , Ferritinas/metabolismo , Ferro/metabolismo , Camundongos , Coativadores de Receptor Nuclear/genética , Fatores de Transcrição/metabolismoRESUMO
We have recently described Pz-1, a benzimidazole-based type-2 RET and VEGFR2 inhibitor. Based on a kinome scan, here we show that Pz-1 is also a potent (IC50 < 1 nM) TRKA/B/C inhibitor. Pz-1 potently inhibited proliferation of human cancer cells carrying either RET- or TRKA oncoproteins (IC50 ~ 1 nM), with a negligible effect against RET- and TRKA-negative cells. By testing mutations, known to mediate resistance to other compounds, RET G810R/S, but not L730I/V, E732K, V738A and Y806N, showed some degree of resistance to Pz-1. In the case of TRKA, G595R and F589L, but not G667C, showed some degree of resistance. In xenograft models, orally administered Pz-1 almost completely inhibited RET- and TRKA-mutant tumours at 1-3 mg/kg/day but showed a reduced effect on RET/TRKA-negative cancer models. The activity, albeit reduced, on RET/TRKA-negative tumours may be justified by VEGFR2 inhibition. Tumours induced by NIH3T3 cells transfected by RET G810R and TRKA G595R featured resistance to Pz-1, demonstrating that RET or TRKA inhibition is critical for its anti-tumourigenic effect. In conclusion, Pz-1 represents a new powerful kinase inhibitor with distinct activity towards cancers induced by oncogenic RET and TRKA variants, including some mutants displaying resistance to other drugs.
Assuntos
Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-ret/metabolismo , Receptor trkA/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Antineoplásicos/farmacologia , Linhagem Celular , Linhagem Celular Tumoral , Células HEK293 , Humanos , Camundongos , Células NIH 3T3 , Neoplasias/metabolismoRESUMO
Anemia in ß-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 ß-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 ß-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 ß-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 ß-thalassemia-related liver iron overload. In ex vivo studies on erythroid precursors from patients with ß-thalassemia, mitapivat enhanced erythropoiesis, promoted erythroid maturation, and decreased apoptosis. Overall, pyruvate kinase activation as a treatment modality for ß-thalassemia in preclinical model systems had multiple beneficial effects in the erythropoietic compartment and beyond, providing a strong scientific basis for further clinical trials.
Assuntos
Ativadores de Enzimas/farmacologia , Hemólise/efeitos dos fármacos , Piperazinas/farmacologia , Piruvato Quinase/metabolismo , Quinolinas/farmacologia , Talassemia beta/tratamento farmacológico , Animais , Modelos Animais de Doenças , Feminino , Camundongos , Camundongos Transgênicos , Talassemia beta/enzimologia , Talassemia beta/genéticaRESUMO
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.
Assuntos
Fusão Gênica , Neoplasias/patologia , Proteínas Proto-Oncogênicas c-ret/genética , Neoplasias da Glândula Tireoide/patologia , Animais , Humanos , Neoplasias/genética , Neoplasias da Glândula Tireoide/genéticaRESUMO
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 in vivo 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 (IC50) 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.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-ret/antagonistas & inibidores , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Descoberta de Drogas , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Mutação , Células NIH 3T3 , Polifarmacologia , Ligação Proteica , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-ret/genética , Proteínas Proto-Oncogênicas c-ret/metabolismo , Transdução de Sinais/efeitos dos fármacos , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
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.
Assuntos
Apoferritinas/metabolismo , Citotoxicidade Imunológica/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Ferro/metabolismo , Células Matadoras Naturais/imunologia , Animais , Apoferritinas/genética , Linhagem Celular Tumoral , Células Cultivadas , Citotoxicidade Imunológica/genética , Desferroxamina/farmacologia , Embrião de Mamíferos/citologia , Fibroblastos/citologia , Fibroblastos/imunologia , Fibroblastos/metabolismo , Expressão Gênica/efeitos dos fármacos , Células HeLa , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Interferon gama/farmacologia , Células K562 , Células Matadoras Naturais/metabolismo , Células MCF-7 , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Coativadores de Receptor Nuclear/genética , Coativadores de Receptor Nuclear/metabolismo , Interferência de RNA , Sideróforos/farmacologiaRESUMO
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.
Assuntos
Senescência Celular , Oncogenes , Neoplasias da Glândula Tireoide/patologia , Animais , Apoptose , Bovinos , Dano ao DNA , Modelos Animais de Doenças , Ativação Enzimática , Feminino , Fatores de Transcrição Forkhead/metabolismo , Hiperplasia , Masculino , Camundongos Transgênicos , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Células Epiteliais da Tireoide/patologia , Glândula Tireoide/patologia , Tireotropina/metabolismo , Tiroxina/metabolismoRESUMO
AIMS: Iron overload (IO) is a life-threatening complication of chronic hemolytic disorders such as ß-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 ß-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 ß-thalassemia. Prx2 is required to support STAT3 transcriptional activity and regulation of Hamp expression. Antioxid. Redox Signal. 28, 1-14.
Assuntos
Eritropoese , Homeostase , Ferro/metabolismo , Peroxirredoxinas/genética , Peroxirredoxinas/metabolismo , Anemia/tratamento farmacológico , Anemia/etiologia , Anemia/metabolismo , Animais , Medula Óssea/metabolismo , Medula Óssea/patologia , Citoproteção/genética , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Hepcidinas/genética , Hepcidinas/metabolismo , Sobrecarga de Ferro/etiologia , Sobrecarga de Ferro/metabolismo , Fígado/metabolismo , Fígado/patologia , Camundongos , Camundongos Knockout , Modelos Biológicos , Estresse Oxidativo , Peroxirredoxinas/farmacologia , Proteínas Recombinantes , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
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 in vitro 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.
RESUMO
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.
Assuntos
Ferro/metabolismo , Coativadores de Receptor Nuclear/metabolismo , Anemia Hipocrômica/metabolismo , Anemia Hipocrômica/patologia , Animais , Autofagia/efeitos dos fármacos , Linhagem Celular , Duodeno/metabolismo , Duodeno/patologia , Eritrócitos/citologia , Eritrócitos/metabolismo , Eritropoese/efeitos dos fármacos , Feminino , Ferritinas/metabolismo , Hepcidinas/metabolismo , Sobrecarga de Ferro/mortalidade , Sobrecarga de Ferro/patologia , Ferro da Dieta/farmacologia , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos Knockout , Coativadores de Receptor Nuclear/química , Coativadores de Receptor Nuclear/genética , Oxirredutases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Baço/metabolismo , Baço/patologia , Regulação para Cima/efeitos dos fármacosRESUMO
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.