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1.
Cell ; 165(4): 867-81, 2016 May 05.
Artículo en Inglés | MEDLINE | ID: mdl-27133164

RESUMEN

Fanconi anemia (FA) pathway genes are important tumor suppressors whose best-characterized function is repair of damaged nuclear DNA. Here, we describe an essential role for FA genes in two forms of selective autophagy. Genetic deletion of Fancc blocks the autophagic clearance of viruses (virophagy) and increases susceptibility to lethal viral encephalitis. Fanconi anemia complementation group C (FANCC) protein interacts with Parkin, is required in vitro and in vivo for clearance of damaged mitochondria, and decreases mitochondrial reactive oxygen species (ROS) production and inflammasome activation. The mitophagy function of FANCC is genetically distinct from its role in genomic DNA damage repair. Moreover, additional genes in the FA pathway, including FANCA, FANCF, FANCL, FANCD2, BRCA1, and BRCA2, are required for mitophagy. Thus, members of the FA pathway represent a previously undescribed class of selective autophagy genes that function in immunity and organellar homeostasis. These findings have implications for understanding the pathogenesis of FA and cancers associated with mutations in FA genes.


Asunto(s)
Proteína del Grupo de Complementación C de la Anemia de Fanconi/metabolismo , Animales , Autofagia , Embrión de Mamíferos/citología , Proteína del Grupo de Complementación C de la Anemia de Fanconi/genética , Proteínas del Grupo de Complementación de la Anemia de Fanconi/metabolismo , Fibroblastos/metabolismo , Células HeLa , Herpesvirus Humano 1/metabolismo , Humanos , Inflamasomas/metabolismo , Ratones , Mitofagia , Especies Reactivas de Oxígeno/metabolismo , Virus Sindbis/metabolismo
2.
Pharmacol Res ; 201: 107092, 2024 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-38311014

RESUMEN

AP endonuclease-1/Redox factor-1 (APE1/Ref-1 or Ref-1) is a multifunctional protein that is overexpressed in most aggressive cancers and impacts various cancer cell signaling pathways. Ref-1's redox activity plays a significant role in activating transcription factors (TFs) such as NFκB, HIF1α, STAT3 and AP-1, which are crucial contributors to the development of tumors and metastatic growth. Therefore, development of potent, selective inhibitors to target Ref-1 redox function is an appealing approach for therapeutic intervention. A first-generation compound, APX3330 successfully completed phase I clinical trial in adults with progressing solid tumors with favorable response rate, pharmacokinetics (PK), and minimal toxicity. These positive results prompted us to develop more potent analogs of APX3330 to effectively target Ref-1 in solid tumors. In this study, we present structure-activity relationship (SAR) identification and validation of lead compounds that exhibit a greater potency and a similar or better safety profile to APX3330. In order to triage and characterize the most potent and on-target second-generation Ref-1 redox inhibitors, we assayed for PK, mouse and human S9 fraction metabolic stability, in silico ADMET properties, ligand-based WaterLOGSY NMR measurements, pharmacodynamic markers, cell viability in multiple cancer cell types, and two distinct 3-dimensional (3D) cell killing assays (Tumor-Microenvironment on a Chip and 3D spheroid). To characterize the effects of Ref-1 inhibition in vivo, global proteomics was used following treatment with the top four analogs. This study identified and characterized more potent inhibitors of Ref-1 redox function (that outperformed APX3330 by 5-10-fold) with PK studies demonstrating efficacious doses for translation to clinic.


Asunto(s)
ADN-(Sitio Apurínico o Apirimidínico) Liasa , Neoplasias , Adulto , Humanos , Animales , Ratones , Inhibidores de la Angiogénesis , Apoptosis , Bioensayo , Neoplasias/tratamiento farmacológico , Microambiente Tumoral
3.
Hum Mol Genet ; 30(17): 1607-1617, 2021 08 12.
Artículo en Inglés | MEDLINE | ID: mdl-34075397

RESUMEN

Neurofibromatosis Type II (NF2) is an autosomal dominant cancer predisposition syndrome in which germline haploinsufficiency at the NF2 gene confers a greatly increased propensity for tumor development arising from tissues of neural crest derived origin. NF2 encodes the tumor suppressor, Merlin, and its biochemical function is incompletely understood. One well-established function of Merlin is as a negative regulator of group A serine/threonine p21-activated kinases (PAKs). In these studies we explore the role of PAK1 and its closely related paralog, PAK2, both pharmacologically and genetically, in Merlin-deficient Schwann cells and in a genetically engineered mouse model (GEMM) that develops spontaneous vestibular and spinal schwannomas. We demonstrate that PAK1 and PAK2 are both hyper activated in Merlin-deficient murine schwannomas. In preclinical trials, a pan Group A PAK inhibitor, FRAX-1036, transiently reduced PAK1 and PAK2 phosphorylation in vitro, but had insignificant efficacy in vivo. NVS-PAK1-1, a PAK1 selective inhibitor, had a greater but still minimal effect on our GEMM phenotype. However, genetic ablation of Pak1 but not Pak2 reduced tumor formation in our NF2 GEMM. Moreover, germline genetic deletion of Pak1 was well tolerated, while conditional deletion of Pak2 in Schwann cells resulted in significant morbidity and mortality. These data support the further development of PAK1-specific small molecule inhibitors and the therapeutic targeting of PAK1 in vestibular schwannomas and argue against PAK1 and PAK2 existing as functionally redundant protein isoforms in Schwann cells.


Asunto(s)
Neurofibromatosis 2/genética , Quinasas p21 Activadas/metabolismo , Animales , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Genes Supresores de Tumor/efectos de los fármacos , Indoles , Longevidad , Ratones , Neurilemoma/genética , Neurofibromatosis 2/metabolismo , Neurofibromina 2/genética , Fosforilación , Piperidinas , Pirimidinas , Células de Schwann/metabolismo , Quinasas p21 Activadas/genética
4.
N Engl J Med ; 382(15): 1430-1442, 2020 04 09.
Artículo en Inglés | MEDLINE | ID: mdl-32187457

RESUMEN

BACKGROUND: No approved therapies exist for inoperable plexiform neurofibromas in patients with neurofibromatosis type 1. METHODS: We conducted an open-label, phase 2 trial of selumetinib to determine the objective response rate among patients with plexiform neurofibromas and to assess clinical benefit. Children with neurofibromatosis type 1 and symptomatic inoperable plexiform neurofibromas received oral selumetinib twice daily at a dose of 25 mg per square meter of body-surface area on a continuous dosing schedule (28-day cycles). Volumetric magnetic resonance imaging and clinical outcome assessments (pain, quality of life, disfigurement, and function) were performed at least every four cycles. Children rated tumor pain intensity on a scale from 0 (no pain) to 10 (worst pain imaginable). RESULTS: A total of 50 children (median age, 10.2 years; range, 3.5 to 17.4) were enrolled from August 2015 through August 2016. The most frequent neurofibroma-related symptoms were disfigurement (44 patients), motor dysfunction (33), and pain (26). A total of 35 patients (70%) had a confirmed partial response as of March 29, 2019, and 28 of these patients had a durable response (lasting ≥1 year). After 1 year of treatment, the mean decrease in child-reported tumor pain-intensity scores was 2 points, considered a clinically meaningful improvement. In addition, clinically meaningful improvements were seen in child-reported and parent-reported interference of pain in daily functioning (38% and 50%, respectively) and overall health-related quality of life (48% and 58%, respectively) as well as in functional outcomes of strength (56% of patients) and range of motion (38% of patients). Five patients discontinued treatment because of toxic effects possibly related to selumetinib, and 6 patients had disease progression. The most frequent toxic effects were nausea, vomiting, or diarrhea; an asymptomatic increase in the creatine phosphokinase level; acneiform rash; and paronychia. CONCLUSIONS: In this phase 2 trial, most children with neurofibromatosis type 1 and inoperable plexiform neurofibromas had durable tumor shrinkage and clinical benefit from selumetinib. (Funded by the Intramural Research Program of the National Institutes of Health and others; ClinicalTrials.gov number, NCT01362803.).


Asunto(s)
Bencimidazoles/uso terapéutico , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Neurofibroma Plexiforme/tratamiento farmacológico , Neurofibromatosis 1/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéutico , Adolescente , Bencimidazoles/efectos adversos , Niño , Preescolar , Femenino , Humanos , Masculino , Náusea/inducido químicamente , Neurofibroma Plexiforme/complicaciones , Neurofibroma Plexiforme/patología , Neurofibromatosis 1/complicaciones , Neurofibromatosis 1/patología , Dolor/etiología , Medición de Resultados Informados por el Paciente , Supervivencia sin Progresión , Inhibidores de Proteínas Quinasas/efectos adversos , Carga Tumoral/efectos de los fármacos
5.
Cell ; 135(3): 437-48, 2008 Oct 31.
Artículo en Inglés | MEDLINE | ID: mdl-18984156

RESUMEN

Interactions between tumorigenic cells and their surrounding microenvironment are critical for tumor progression yet remain incompletely understood. Germline mutations in the NF1 tumor suppressor gene cause neurofibromatosis type 1 (NF1), a common genetic disorder characterized by complex tumors called neurofibromas. Genetic studies indicate that biallelic loss of Nf1 is required in the tumorigenic cell of origin in the embryonic Schwann cell lineage. However, in the physiologic state, Schwann cell loss of heterozygosity is not sufficient for neurofibroma formation and Nf1 haploinsufficiency in at least one additional nonneoplastic lineage is required for tumor progression. Here, we establish that Nf1 heterozygosity of bone marrow-derived cells in the tumor microenvironment is sufficient to allow neurofibroma progression in the context of Schwann cell Nf1 deficiency. Further, genetic or pharmacologic attenuation of c-kit signaling in Nf1+/- hematopoietic cells diminishes neurofibroma initiation and progression. Finally, these studies implicate mast cells as critical mediators of tumor initiation.


Asunto(s)
Neurofibroma/metabolismo , Neurofibromina 1/metabolismo , Proteínas Proto-Oncogénicas c-kit/metabolismo , Animales , Benzamidas , Médula Ósea/fisiopatología , Trasplante de Médula Ósea , Preescolar , Genes de Neurofibromatosis 1 , Humanos , Mesilato de Imatinib , Mastocitos/metabolismo , Ratones , Ratones Endogámicos C57BL , Neurofibroma/tratamiento farmacológico , Neurofibroma/genética , Neurofibroma/patología , Neurofibroma Plexiforme/tratamiento farmacológico , Neurofibroma Plexiforme/metabolismo , Piperazinas/uso terapéutico , Pirimidinas/uso terapéutico , Células de Schwann/metabolismo
6.
J Biol Chem ; 295(29): 9948-9958, 2020 07 17.
Artículo en Inglés | MEDLINE | ID: mdl-32471868

RESUMEN

Neurofibromatosis type 1 (NF1) is a common cancer predisposition syndrome caused by mutations in the NF1 tumor suppressor gene. NF1 encodes neurofibromin, a GTPase-activating protein for RAS proto-oncogene GTPase (RAS). Plexiform neurofibromas are a hallmark of NF1 and result from loss of heterozygosity of NF1 in Schwann cells, leading to constitutively activated p21RAS. Given the inability to target p21RAS directly, here we performed an shRNA library screen of all human kinases and Rho-GTPases in a patient-derived NF1-/- Schwann cell line to identify novel therapeutic targets to disrupt PN formation and progression. Rho family members, including Rac family small GTPase 1 (RAC1), were identified as candidates. Corroborating these findings, we observed that shRNA-mediated knockdown of RAC1 reduces cell proliferation and phosphorylation of extracellular signal-regulated kinase (ERK) in NF1-/- Schwann cells. Genetically engineered Nf1flox/flox;PostnCre+ mice, which develop multiple PNs, also exhibited increased RAC1-GTP and phospho-ERK levels compared with Nf1flox/flox;PostnCre- littermates. Notably, mice in which both Nf1 and Rac1 loci were disrupted (Nf1flox/floxRac1flox/flox;PostnCre+) were completely free of tumors and had normal phospho-ERK activity compared with Nf1flox/flox ;PostnCre+ mice. We conclude that the RAC1-GTPase is a key downstream node of RAS and that genetic disruption of the Rac1 allele completely prevents PN tumor formation in vivo in mice.


Asunto(s)
Técnicas de Silenciamiento del Gen , Neoplasias Primarias Secundarias , Neurofibroma Plexiforme , Neurofibromatosis 1 , Neuropéptidos/deficiencia , Proteína de Unión al GTP rac1/deficiencia , Animales , Ratones , Ratones Noqueados , Neoplasias Primarias Secundarias/enzimología , Neoplasias Primarias Secundarias/genética , Neoplasias Primarias Secundarias/patología , Neoplasias Primarias Secundarias/prevención & control , Neurofibroma Plexiforme/enzimología , Neurofibroma Plexiforme/genética , Neurofibroma Plexiforme/prevención & control , Neurofibromatosis 1/enzimología , Neurofibromatosis 1/genética , Neurofibromatosis 1/patología , Neurofibromina 1/deficiencia , Neurofibromina 1/metabolismo , Neuropéptidos/metabolismo , Proto-Oncogenes Mas , Proteína de Unión al GTP rac1/metabolismo
7.
Hum Mol Genet ; 28(16): 2752-2762, 2019 08 15.
Artículo en Inglés | MEDLINE | ID: mdl-31091306

RESUMEN

Plexiform neurofibroma (PN) tumors are a hallmark manifestation of neurofibromatosis type 1 (NF1) that arise in the Schwann cell (SC) lineage. NF1 is a common heritable cancer predisposition syndrome caused by germline mutations in the NF1 tumor suppressor, which encodes a GTPase-activating protein called neurofibromin that negatively regulates Ras proteins. Whereas most PN are clinically indolent, a subset progress to atypical neurofibromatous neoplasms of uncertain biologic potential (ANNUBP) and/or to malignant peripheral nerve sheath tumors (MPNSTs). In small clinical series, loss of 9p21.3, which includes the CDKN2A locus, has been associated with the genesis of ANNUBP. Here we show that the Cdkn2a alternate reading frame (Arf) serves as a gatekeeper tumor suppressor in mice that prevents PN progression by inducing senescence-mediated growth arrest in aberrantly proliferating Nf1-/- SC. Conditional ablation of Nf1 and Arf in the neural crest-derived SC lineage allows escape from senescence, resulting in tumors that accurately phenocopy human ANNUBP and progress to MPNST with high penetrance. This animal model will serve as a platform to study the clonal development of ANNUBP and MPNST and to identify new therapies to treat existing tumors and to prevent disease progression.


Asunto(s)
Transformación Celular Neoplásica/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/deficiencia , Neurofibroma/genética , Neurofibroma/patología , Neurofibromatosis 1/genética , Animales , Biomarcadores de Tumor , Línea Celular Tumoral , Transformación Celular Neoplásica/metabolismo , Senescencia Celular/genética , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Genotipo , Xenoinjertos , Humanos , Inmunohistoquímica , Ratones , Mutación , Neoplasias de la Vaina del Nervio/genética , Neoplasias de la Vaina del Nervio/metabolismo , Neoplasias de la Vaina del Nervio/patología , Neurofibroma/metabolismo , Neurofibroma/mortalidad , Neurofibromatosis 1/metabolismo , Células de Schwann/metabolismo , Células de Schwann/patología , Proteínas ras/metabolismo
8.
Br J Cancer ; 124(9): 1566-1580, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33658640

RESUMEN

BACKGROUND: MPNST is a rare soft-tissue sarcoma that can arise from patients with NF1. Existing chemotherapeutic and targeted agents have been unsuccessful in MPNST treatment, and recent findings implicate STAT3 and HIF1-α in driving MPNST. The DNA-binding and transcriptional activity of both STAT3 and HIF1-α is regulated by Redox factor-1 (Ref-1) redox function. A first-generation Ref-1 inhibitor, APX3330, is being tested in cancer clinical trials and could be applied to MPNST. METHODS: We characterised Ref-1 and p-STAT3 expression in various MPNST models. Tumour growth, as well as biomarkers of apoptosis and signalling pathways, were measured by qPCR and western blot following treatment with inhibitors of Ref-1 or STAT3. RESULTS: MPNSTs from Nf1-Arfflox/floxPostnCre mice exhibit significantly increased positivity of p-STAT3 and Ref-1 expression when malignant transformation occurs. Inhibition of Ref-1 or STAT3 impairs MPNST growth in vitro and in vivo and induces apoptosis. Genes highly expressed in MPNST patients are downregulated following inhibition of Ref-1 or STAT3. Several biomarkers downstream of Ref-1 or STAT3 were also downregulated following Ref-1 or STAT3 inhibition. CONCLUSIONS: Our findings implicate a unique therapeutic approach to target important MPNST signalling nodes in sarcomas using new first-in-class small molecules for potential translation to the clinic.


Asunto(s)
Biomarcadores de Tumor/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Regulación Neoplásica de la Expresión Génica , Neurofibrosarcoma/patología , Factor de Transcripción STAT3/metabolismo , Adolescente , Animales , Apoptosis , Biomarcadores de Tumor/genética , Proliferación Celular , ADN-(Sitio Apurínico o Apirimidínico) Liasa/genética , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Neurofibrosarcoma/genética , Neurofibrosarcoma/metabolismo , Pronóstico , Factor de Transcripción STAT3/genética , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto
9.
Blood Cells Mol Dis ; 86: 102492, 2021 02.
Artículo en Inglés | MEDLINE | ID: mdl-32896825

RESUMEN

Fanconi anemia (FA) is associated with bone marrow failure. Bone marrow (BM) from patients with FA and fanca-/- and fancc-/- mice are deficient in hematopoietic stem (HSCs) and progenitor cells (HPCs). Decreased HSCs/HPCs compromise their use in human and mouse hematopoietic cell transplantation (HCT) and gene therapy to correct genetic defects causing FA. We reported increased collection of HSCs from mouse bone marrow and mobilized peripheral blood, and human cord blood of normal donors after collection/processing in low (3%) oxygen (physioxia). We assessed comparative contents of long-term (LT)-HSCs from BM of fanca-/- and fancc-/- when collected/processed at 3% O2, in order to negate effects of extra physiological shock stress (EPHOSS) induced by collection/processing in ambient air. Collection/processing of BM from fanca-/- and fancc-/- mice in physioxia demonstrated a ≥3-fold increase in LT-HSCs compared to that in ambient air. This was associated with decreased phenotypic multipotential progenitor cells and functional granulocyte macrophage, erythroid, and multi-potential progenitors, results similar to that for BM from normal donor mice. Increased collection of HSCs could have clinical applicability for gene therapy and HCT.


Asunto(s)
Células de la Médula Ósea/citología , Proteína del Grupo de Complementación A de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación C de la Anemia de Fanconi/genética , Células Madre Hematopoyéticas/citología , Animales , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/patología , Recuento de Células , Hipoxia de la Célula , Separación Celular , Células Cultivadas , Anemia de Fanconi/genética , Anemia de Fanconi/patología , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/patología , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados
10.
Genet Med ; 22(11): 1786-1793, 2020 11.
Artículo en Inglés | MEDLINE | ID: mdl-32601387

RESUMEN

PURPOSE: Plexiform neurofibromas (pNF) develop in children with neurofibromatosis type 1 (NF1) and can be associated with several skeletal comorbidities. Preclinical mouse studies revealed Nf1 deficiency in osteoprogenitor cells disrupts, in a MEK-dependent manner, pyrophosphate (PPi) homeostasis and skeletal mineralization. The etiology of NF-associated skeletal manifestations remains unknown. METHODS: We used mouse models of NF1 neurofibromas to assess bone mineralization of skeletal structures adjacent to tumors. Expression of genes involved in pyrophosphate homeostasis was assessed in mouse and human NF tumors and Schwann cell cultures. We used dual-energy X-ray absorptiometry (DXA) to assess tumor-associated changes in bone mineral density (BMD) in an individual with NF1 following treatment with the MEK inhibitor selumetinib. RESULTS: We detected increased nonmineralized bone surfaces adjacent to tumors in mouse models of NF1 neurofibromas. Expression of Enpp1, a PPi-generating ectophosphatase, and ANKH, a PPi transporter, was increased in mouse and human neurofibroma-derived tissues and Schwann cells, respectively. In one patient, tumor-associated reductions in BMD were partially rescued following therapy with selumetinib. CONCLUSION: Results indicate that NF-associated skeletal pathologies in NF1 are associated with dysregulated pyrophosphate homeostasis in adjacent NF tumors and suggest that treatment of NFs with MEK inhibitors may improve skeletal manifestations of the disease.


Asunto(s)
Neurofibroma Plexiforme , Neurofibroma , Neurofibromatosis 1 , Animales , Humanos , Ratones , Neurofibroma Plexiforme/genética , Neurofibromatosis 1/genética , Inhibidores de Proteínas Quinasas , Células de Schwann
11.
Pediatr Blood Cancer ; 67(8): e28372, 2020 08.
Artículo en Inglés | MEDLINE | ID: mdl-32459399

RESUMEN

BACKGROUND: Neurofibromatosis type 1 (NF1) is a common genetic disorder characterized by plexiform neurofibromas (pNF), which are thought to be congenital tumors that arise in utero and enlarge throughout life. Genetic studies in murine models delineated an indispensable role for the stem cell factor (SCF)/c-kit pathway in pNF initiation and progression. A subsequent phase 2 clinical trial using imatinib mesylate to inhibit SCF/c-kit demonstrated tumor shrinkage in a subset of preexisting pNF; however, imatinib's role on preventing pNF development has yet to be explored. PROCEDURE: We evaluated the effect of imatinib dosed at 10-100 mg/kg/day for 12 weeks to one-month-old Nf1flox/flox ;PostnCre(+) mice, prior to onset of pNF formation. To determine durability of response, we then monitored for pNF growth at later time points, comparing imatinib- with vehicle-treated mice. We assessed gross and histopathological analysis of tumor burden. RESULTS: Imatinib administered preventatively led to a significant decrease in pNF number, even at doses as low as 10 mg/kg/day. Tumor development continued to be significantly inhibited after cessation of imatinib dosed at 50 and 100 mg/kg/day. In the cohort of treated mice that underwent prolonged follow-up, the size of residual tumors was significantly reduced as compared with age-matched littermates that received vehicle control. CONCLUSIONS: Early administration of imatinib inhibits pNF genesis in vivo, and effects are sustained after discontinuation of therapy. These findings may guide clinical use of imatinib in young NF1 patients prior to the substantial development of pNF.


Asunto(s)
Mesilato de Imatinib/administración & dosificación , Neoplasias Experimentales/prevención & control , Neurofibroma Plexiforme/prevención & control , Neurofibromatosis 1/prevención & control , Animales , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Ratones , Ratones Transgénicos , Neoplasias Experimentales/genética , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Neurofibroma Plexiforme/genética , Neurofibroma Plexiforme/metabolismo , Neurofibroma Plexiforme/patología , Neurofibromatosis 1/genética , Neurofibromatosis 1/metabolismo , Neurofibromatosis 1/patología
12.
Hum Mol Genet ; 25(6): 1129-39, 2016 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-26740548

RESUMEN

Persons with neurofibromatosis type 1 (NF1) have a predisposition for premature and severe arterial stenosis. Mutations in the NF1 gene result in decreased expression of neurofibromin, a negative regulator of p21(Ras), and increases Ras signaling. Heterozygous Nf1 (Nf1(+/-)) mice develop a marked arterial stenosis characterized by proliferating smooth muscle cells (SMCs) and a predominance of infiltrating macrophages, which closely resembles arterial lesions from NF1 patients. Interestingly, lineage-restricted inactivation of a single Nf1 allele in monocytes/macrophages is sufficient to recapitulate the phenotype observed in Nf1(+/-) mice and to mobilize proinflammatory CCR2+ monocytes into the peripheral blood. Therefore, we hypothesized that CCR2 receptor activation by its primary ligand monocyte chemotactic protein-1 (MCP-1) is critical for monocyte infiltration into the arterial wall and neointima formation in Nf1(+/-) mice. MCP-1 induces a dose-responsive increase in Nf1(+/-) macrophage migration and proliferation that corresponds with activation of multiple Ras kinases. In addition, Nf1(+/-) SMCs, which express CCR2, demonstrate an enhanced proliferative response to MCP-1 when compared with WT SMCs. To interrogate the role of CCR2 activation on Nf1(+/-) neointima formation, we induced neointima formation by carotid artery ligation in Nf1(+/-) and WT mice with genetic deletion of either MCP1 or CCR2. Loss of MCP-1 or CCR2 expression effectively inhibited Nf1(+/-) neointima formation and reduced macrophage content in the arterial wall. Finally, administration of a CCR2 antagonist significantly reduced Nf1(+/-) neointima formation. These studies identify MCP-1 as a potent chemokine for Nf1(+/-) monocytes/macrophages and CCR2 as a viable therapeutic target for NF1 arterial stenosis.


Asunto(s)
Macrófagos/patología , Monocitos/patología , Neointima/patología , Neurofibromatosis 1/patología , Receptores CCR2/metabolismo , Animales , Arterias Carótidas/metabolismo , Arterias Carótidas/patología , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Genes de Neurofibromatosis 1 , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Monocitos/metabolismo , Miocitos del Músculo Liso/metabolismo , Neointima/genética , Neointima/metabolismo , Neurofibromatosis 1/genética , Neurofibromatosis 1/metabolismo , Neurofibromina 1/genética , Receptores CCR2/antagonistas & inhibidores , Receptores CCR2/genética , Transducción de Señal
15.
Haematologica ; 102(6): 1017-1027, 2017 06.
Artículo en Inglés | MEDLINE | ID: mdl-28341737

RESUMEN

Fanconi anemia is a complex heterogeneous genetic disorder with a high incidence of bone marrow failure, clonal evolution to acute myeloid leukemia and mesenchymal-derived congenital anomalies. Increasing evidence in Fanconi anemia and other genetic disorders points towards an interdependence of skeletal and hematopoietic development, yet the impact of the marrow microenvironment in the pathogenesis of the bone marrow failure in Fanconi anemia remains unclear. Here we demonstrated that mice with double knockout of both Fancc and Fancg genes had decreased bone formation at least partially due to impaired osteoblast differentiation from mesenchymal stem/progenitor cells. Mesenchymal stem/progenitor cells from the double knockout mice showed impaired hematopoietic supportive activity. Mesenchymal stem/progenitor cells of patients with Fanconi anemia exhibited similar cellular deficits, including increased senescence, reduced proliferation, impaired osteoblast differentiation and defective hematopoietic stem/progenitor cell supportive activity. Collectively, these studies provide unique insights into the physiological significance of mesenchymal stem/progenitor cells in supporting the marrow microenvironment, which is potentially of broad relevance in hematopoietic stem cell transplantation.


Asunto(s)
Médula Ósea/patología , Microambiente Celular , Anemia de Fanconi/patología , Animales , Huesos/anomalías , Huesos/fisiopatología , Linaje de la Célula , Anemia de Fanconi/fisiopatología , Proteína del Grupo de Complementación C de la Anemia de Fanconi/genética , Hematopoyesis , Trasplante de Células Madre Hematopoyéticas , Células Madre Mesenquimatosas/patología , Ratones , Ratones Noqueados
17.
Acta Neuropathol ; 132(2): 289-307, 2016 08.
Artículo en Inglés | MEDLINE | ID: mdl-27236462

RESUMEN

Schwannomas are predominantly benign nerve sheath neoplasms caused by Nf2 gene inactivation. Presently, treatment options are mainly limited to surgical tumor resection due to the lack of effective pharmacological drugs. Although the mechanistic understanding of Nf2 gene function has advanced, it has so far been primarily restricted to Schwann cell-intrinsic events. Extracellular cues determining Schwann cell behavior with regard to schwannoma development remain unknown. Here we show pro-tumourigenic microenvironmental effects on Schwann cells where an altered axonal microenvironment in cooperation with injury signals contribute to a persistent regenerative Schwann cell response promoting schwannoma development. Specifically in genetically engineered mice following crush injuries on sciatic nerves, we found macroscopic nerve swellings in mice with homozygous nf2 gene deletion in Schwann cells and in animals with heterozygous nf2 knockout in both Schwann cells and axons. However, patient-mimicking schwannomas could only be provoked in animals with combined heterozygous nf2 knockout in Schwann cells and axons. We identified a severe re-myelination defect and sustained macrophage presence in the tumor tissue as major abnormalities. Strikingly, treatment of tumor-developing mice after nerve crush injury with medium-dose aspirin significantly decreased schwannoma progression in this disease model. Our results suggest a multifactorial concept for schwannoma formation-emphasizing axonal factors and mechanical nerve irritation as predilection site for schwannoma development. Furthermore, we provide evidence supporting the potential efficacy of anti-inflammatory drugs in the treatment of schwannomas.


Asunto(s)
Axones/patología , Neurilemoma/patología , Células de Schwann/patología , Nervio Ciático/patología , Microambiente Tumoral/fisiología , Animales , Ratones Transgénicos , Vaina de Mielina/patología , Neurilemoma/genética , Neurofibromatosis 2/genética , Microambiente Tumoral/genética
18.
Stem Cells ; 33(5): 1630-41, 2015 May.
Artículo en Inglés | MEDLINE | ID: mdl-25586960

RESUMEN

p21-Activated kinase 2 (Pak2), a serine/threonine kinase, has been previously shown to be essential for hematopoietic stem cell (HSC) engraftment. However, Pak2 modulation of long-term hematopoiesis and lineage commitment remain unreported. Using a conditional Pak2 knockout mouse model, we found that disruption of Pak2 in HSCs induced profound leukopenia and a mild macrocytic anemia. Although loss of Pak2 in HSCs leads to less efficient short- and long-term competitive hematopoiesis than wild-type cells, it does not affect HSC self-renewal per se. Pak2 disruption decreased the survival and proliferation of multicytokine stimulated immature progenitors. Loss of Pak2 skewed lineage differentiation toward granulocytopoiesis and monocytopoiesis in mice as evidenced by (a) a three- to sixfold increase in the percentage of peripheral blood granulocytes and a significant increase in the percentage of granulocyte-monocyte progenitors in mice transplanted with Pak2-disrupted bone marrow (BM); (b)Pak2-disrupted BM and c-kit(+) cells yielded higher numbers of more mature subsets of granulocyte-monocyte colonies and polymorphonuclear neutrophils, respectively, when cultured in the presence of granulocyte-macrophage colony-stimulating factor. Pak2 disruption resulted, respectively, in decreased and increased gene expression of transcription factors JunB and c-Myc, which may suggest underlying mechanisms by which Pak2 regulates granulocyte-monocyte lineage commitment. Furthermore, Pak2 disruption led to (a) higher percentage of CD4(+) CD8(+) double positive T cells and lower percentages of CD4(+) CD8(-) or CD4(-) CD8(+) single positive T cells in thymus and (b) decreased numbers of mature B cells and increased numbers of Pre-Pro B cells in BM, suggesting defects in lymphopoiesis.


Asunto(s)
Diferenciación Celular , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/enzimología , Quinasas p21 Activadas/metabolismo , Anemia Macrocítica/patología , Animales , Apoptosis , Proliferación Celular , Supervivencia Celular , Eliminación de Gen , Regulación de la Expresión Génica , Hematopoyesis , Leucopenia/patología , Linfopoyesis , Ratones Noqueados , Células Mieloides/patología , Fenotipo , Factores de Transcripción/metabolismo , Quinasas p21 Activadas/deficiencia
19.
Pediatr Blood Cancer ; 63(2): 206-13, 2016 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-26375012

RESUMEN

PURPOSE: Plexiform neurofibromas (pNF) are pathognomonic nerve and soft tissue tumors of neurofibromatosis type I (NF1), which are highly resistant to conventional chemotherapy and associated with significant morbidity/mortality. Disruption of aberrant SCF/c-Kit signaling emanating from the pNF microenvironment induced the first ever objective therapeutic responses in a recent phase 2 trial. Sunitinib malate is a potent, highly selective RTK inhibitor with activity against c-Kit, PDGFR, and VEGFR, which have also been implicated in the pathogenesis of these lesions. Here, we evaluate the efficacy of sunitinib malate in a preclinical Krox20;Nf1(flox/-) pNF murine model. EXPERIMENTAL DESIGN: Proliferation, ß-hexosaminidase release (degranulation), and Erk1/2 phosphorylation were assessed in sunitinib treated Nf1(+/-) mast cells and fibroblasts, respectively. Krox20;Nf1(flox/-) mice with established pNF were treated sunitinib or PBS-vehicle control for a duration of 12 weeks. pNF metabolic activity was monitored by serial [(18)F]DG-PET/CT imaging. RESULTS: Sunitinib suppressed multiple in vitro gain-in-functions of Nf1(+/-) mast cells and fibroblasts and attenuated Erk1/2 phosphorylation. Sunitinib treated Krox20;Nf1(flox/-) mice exhibited significant reductions in pNF size, tumor number, and FDG uptake compared to control mice. Histopathology revealed reduced tumor cellularity and infiltrating mast cells, markedly diminished collagen deposition, and increased cellular apoptosis in sunitinib treated pNF. CONCLUSIONS: Collectively, these results demonstrate the efficacy of sunitinib in reducing tumor burden in Krox20;Nf1(flox/-) mice. These preclinical findings demonstrate the utility of inhibiting multiple RTKs in pNF and provide insights into the design of future clinical trials.


Asunto(s)
Antineoplásicos/farmacología , Indoles/farmacología , Neurofibroma Plexiforme/patología , Pirroles/farmacología , Microambiente Tumoral/efectos de los fármacos , Animales , Western Blotting , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/patología , Masculino , Mastocitos/efectos de los fármacos , Mastocitos/patología , Ratones , Ratones Transgénicos , Tomografía de Emisión de Positrones , Sunitinib
20.
Circulation ; 129(11): 1213-24, 2014 Mar 18.
Artículo en Inglés | MEDLINE | ID: mdl-24370551

RESUMEN

BACKGROUND: Neurofibromatosis type 1 (NF1) is a genetic disorder resulting from mutations in the NF1 tumor suppressor gene. Neurofibromin, the protein product of NF1, functions as a negative regulator of Ras activity in circulating hematopoietic and vascular wall cells, which are critical for maintaining vessel wall homeostasis. NF1 patients have evidence of chronic inflammation resulting in the development of premature cardiovascular disease, including arterial aneurysms, which may manifest as sudden death. However, the molecular pathogenesis of NF1 aneurysm formation is unknown. METHOD AND RESULTS: With the use of an angiotensin II-induced aneurysm model, we demonstrate that heterozygous inactivation of Nf1 (Nf1(+/-)) enhanced aneurysm formation with myeloid cell infiltration and increased oxidative stress in the vessel wall. Using lineage-restricted transgenic mice, we show that loss of a single Nf1 allele in myeloid cells is sufficient to recapitulate the Nf1(+/-) aneurysm phenotype in vivo. Finally, oral administration of simvastatin or the antioxidant apocynin reduced aneurysm formation in Nf1(+/-) mice. CONCLUSION: These data provide genetic and pharmacological evidence that Nf1(+/-) myeloid cells are the cellular triggers for aneurysm formation in a novel model of NF1 vasculopathy and provide a potential therapeutic target.


Asunto(s)
Aneurisma/metabolismo , Células Mieloides/metabolismo , Neurofibromina 1/deficiencia , Aneurisma/tratamiento farmacológico , Aneurisma/genética , Animales , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Transgénicos , Neurofibromina 1/genética , Simvastatina/uso terapéutico
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