RESUMEN
Immune cells need to sustain a state of constant alertness over a lifetime. Yet, little is known about the regulatory processes that control the fluent and fragile balance that is called homeostasis. Here we demonstrate that JAK-STAT signaling, beyond its role in immune responses, is a major regulator of immune cell homeostasis. We investigated JAK-STAT-mediated transcription and chromatin accessibility across 12 mouse models, including knockouts of all STAT transcription factors and of the TYK2 kinase. Baseline JAK-STAT signaling was detected in CD8+ T cells and macrophages of unperturbed mice-but abrogated in the knockouts and in unstimulated immune cells deprived of their normal tissue context. We observed diverse gene-regulatory programs, including effects of STAT2 and IRF9 that were independent of STAT1. In summary, our large-scale dataset and integrative analysis of JAK-STAT mutant and wild-type mice uncovered a crucial role of JAK-STAT signaling in unstimulated immune cells, where it contributes to a poised epigenetic and transcriptional state and helps prepare these cells for rapid response to immune stimuli.
Asunto(s)
Homeostasis , Quinasas Janus , Macrófagos , Ratones Noqueados , Factores de Transcripción STAT , Transducción de Señal , Animales , Ratones , Macrófagos/inmunología , Macrófagos/metabolismo , Quinasas Janus/metabolismo , Factores de Transcripción STAT/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT1/genética , Ratones Endogámicos C57BL , Subunidad gamma del Factor 3 de Genes Estimulados por el Interferón/metabolismo , Subunidad gamma del Factor 3 de Genes Estimulados por el Interferón/genética , TYK2 Quinasa/metabolismo , TYK2 Quinasa/genética , Regulación de la Expresión GénicaRESUMEN
ABSTRACT: Patients with T- and natural killer (NK)-cell neoplasms frequently have somatic STAT5B gain-of-function mutations. The most frequent STAT5B mutation is STAT5BN642H, which is known to drive murine T-cell leukemia, although its role in NK-cell malignancies is unclear. Introduction of the STAT5BN642H mutation into human NK-cell lines enhances their potential to induce leukemia in mice. We have generated a mouse model that enables tissue-specific expression of STAT5BN642H and have selectively expressed the mutated STAT5B in hematopoietic cells (N642Hvav/+) or exclusively in NK cells (N642HNK/NK). All N642Hvav/+ mice rapidly develop an aggressive T/NKT-cell leukemia, whereas N642HNK/NK mice display an indolent NK-large granular lymphocytic leukemia (NK-LGLL) that progresses to an aggressive leukemia with age. Samples from patients with NK-cell leukemia have a distinctive transcriptional signature driven by mutant STAT5B, which overlaps with that of murine leukemic N642HNK/NK NK cells. To our knowledge, we have generated the first reliable STAT5BN642H-driven preclinical mouse model that displays an indolent NK-LGLL progressing to aggressive NK-cell leukemia. This novel in vivo tool will enable us to explore the transition from an indolent to an aggressive disease and will thus permit the study of prevention and treatment options for NK-cell malignancies.
Asunto(s)
Células Asesinas Naturales , Leucemia Linfocítica Granular Grande , Factor de Transcripción STAT5 , Animales , Factor de Transcripción STAT5/genética , Factor de Transcripción STAT5/metabolismo , Ratones , Células Asesinas Naturales/metabolismo , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/patología , Humanos , Leucemia Linfocítica Granular Grande/genética , Leucemia Linfocítica Granular Grande/patología , Modelos Animales de Enfermedad , Linaje de la Célula/genética , Mutación , Ratones TransgénicosRESUMEN
Gain-of-function mutations in the signal transducer and activator of transcription 3 (STAT3) gene are recurrently identified in patients with large granular lymphocytic leukemia (LGLL) and in some cases of natural killer (NK)/T-cell and adult T-cell leukemia/lymphoma. To understand the consequences and molecular mechanisms contributing to disease development and oncogenic transformation, we developed murine hematopoietic stem and progenitor cell models that express mutated STAT3Y640F. These cells show accelerated proliferation and enhanced self-renewal potential. We integrated gene expression analyses and chromatin occupancy profiling of STAT3Y640F-transformed cells with data from patients with T-LGLL. This approach uncovered a conserved set of direct transcriptional targets of STAT3Y640F. Among these, strawberry notch homolog 2 (SBNO2) represents an essential transcriptional target, which was identified by a comparative genome-wide CRISPR/Cas9-based loss-of-function screen. The STAT3-SBNO2 axis is also present in NK-cell leukemia, T-cell non-Hodgkin lymphoma, and NPM-ALK-rearranged T-cell anaplastic large cell lymphoma (T-ALCL), which are driven by STAT3-hyperactivation/mutation. In patients with NPM-ALK+ T-ALCL, high SBNO2 expression correlates with shorter relapse-free and overall survival. Our findings identify SBNO2 as a potential therapeutic intervention site for STAT3-driven hematopoietic malignancies.
Asunto(s)
Neoplasias Hematológicas , Factor de Transcripción STAT3 , Animales , Humanos , Ratones , Quinasa de Linfoma Anaplásico/metabolismo , Línea Celular Tumoral , Neoplasias Hematológicas/genética , Linfoma Anaplásico de Células Grandes/genética , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismoRESUMEN
The transcription factors signal transducer and activator of transcription 5A (STAT5A) and STAT5B are critical in hematopoiesis and leukemia. They are widely believed to have redundant functions, but we describe a unique role for STAT5B in driving the self-renewal of hematopoietic and leukemic stem cells (HSCs/LSCs). We find STAT5B to be specifically activated in HSCs and LSCs, where it induces many genes associated with quiescence and self-renewal, including the surface marker CD9. Levels of CD9 represent a prognostic marker for patients with STAT5-driven leukemia, and our findings suggest that anti-CD9 antibodies may be useful in their treatment to target and eliminate LSCs. We show that it is vital to consider STAT5A and STAT5B as distinct entities in normal and malignant hematopoiesis.
Asunto(s)
Células Madre Hematopoyéticas/patología , Leucemia/patología , Células Madre Neoplásicas/patología , Factor de Transcripción STAT5/metabolismo , Transducción de Señal , Tetraspanina 29/metabolismo , Animales , Autorrenovación de las Células , Hematopoyesis , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Humanos , Leucemia/metabolismo , Ratones , Ratones Endogámicos C57BL , Células Madre Neoplásicas/citología , Células Madre Neoplásicas/metabolismo , Células Tumorales CultivadasRESUMEN
Tyrosine kinase 2 (TYK2) is a member of the Janus kinase/signal transducer and activator of transcription pathway, which is central in cytokine signaling. Previously, germline TYK2 mutations have been described in two patients developing de novo T-cell acute lymphoblastic leukemias (T-ALL) or precursor B-ALL. The mutations (P760L and G761V) are located within the regulatory pseudokinase domain and lead to constitutive activation of TYK2. We demonstrate the transformation capacity of TYK2 P760L in hematopoietic cell systems including primary bone marrow cells. In vivo engraftment of TYK2 P760L-expressing cell lines led to development of leukemia. A kinase inhibitor screen uncovered that oncogenic TYK2 acts synergistically with the PI3K/AKT/mTOR and CDK4/6 pathways. Accordingly, the TYK2-specific inhibitor deucravacitinib (BMS986165) reduces cell viability of TYK2 P760L-transformed cell models and ex vivo cultured TYK2 P760L-mutated patient- derived xenograft cells most efficiently when combined with mTOR or CDK4/6 inhibitors. Our study thereby pioneers novel treatment options for patients suffering from TYK2-driven acute leukemia.
Asunto(s)
Leucemia-Linfoma Linfoblástico de Células T Precursoras , TYK2 Quinasa , Humanos , Línea Celular , Quinasa 4 Dependiente de la Ciclina , Fosfatidilinositol 3-Quinasas , Serina-Treonina Quinasas TOR , TYK2 Quinasa/genética , TYK2 Quinasa/metabolismoRESUMEN
Tissue-resident macrophages are of vital importance as they preserve tissue homeostasis in all mammalian organs. Nevertheless, appropriate cell culture models are still limited. Here, we propose a novel culture model to study and expand murine primary alveolar macrophages (AMs), the tissue-resident macrophages of the lung, in vitro over several months. By providing a combination of granulocyte-macrophage colony-stimulating factor, TGFß, and the PPARγ activator rosiglitazone, we maintain and expand mouse ex vivo cultured AMs (mexAMs) over several months. MexAMs maintain typical morphologic features and stably express primary AM surface markers throughout in vitro culture. They respond to microbial ligands and exhibit an AM-like transcriptional profile, including the expression of AM-specific transcription factors. Furthermore, when transferred into AM-deficient mice, mexAMs efficiently engraft in the lung and fulfill key macrophage functions, leading to a significantly reduced surfactant load in those mice. Altogether, mexAMs provide a novel, simple, and versatile tool to study AM behavior in homeostasis and disease settings.
Asunto(s)
Macrófagos Alveolares/metabolismo , Animales , Animales Recién Nacidos , Células Cultivadas , Modelos Animales de Enfermedad , Hígado/metabolismo , Pulmón/patología , Pulmón/fisiopatología , Macrófagos Alveolares/patología , Ratones Endogámicos C57BL , Fenotipo , Proteinosis Alveolar Pulmonar/metabolismo , Proteinosis Alveolar Pulmonar/patología , Proteinosis Alveolar Pulmonar/fisiopatología , Transcripción GenéticaRESUMEN
Fusion proteins involving Nucleoporin 98 (NUP98) are recurrently found in acute myeloid leukemia (AML) and are associated with poor prognosis. Lack of mechanistic insight into NUP98-fusion-dependent oncogenic transformation has so far precluded the development of rational targeted therapies. We reasoned that different NUP98-fusion proteins deregulate a common set of transcriptional targets that might be exploitable for therapy. To decipher transcriptional programs controlled by diverse NUP98-fusion proteins, we developed mouse models for regulatable expression of NUP98/NSD1, NUP98/JARID1A, and NUP98/DDX10. By integrating chromatin occupancy profiles of NUP98-fusion proteins with transcriptome profiling upon acute fusion protein inactivation in vivo, we defined the core set of direct transcriptional targets of NUP98-fusion proteins. Among those, CDK6 was highly expressed in murine and human AML samples. Loss of CDK6 severely attenuated NUP98-fusion-driven leukemogenesis, and NUP98-fusion AML was sensitive to pharmacologic CDK6 inhibition in vitro and in vivo. These findings identify CDK6 as a conserved, critical direct target of NUP98-fusion proteins, proposing CDK4/CDK6 inhibitors as a new rational treatment option for AML patients with NUP98-fusions.
Asunto(s)
Quinasa 6 Dependiente de la Ciclina/metabolismo , Sistemas de Liberación de Medicamentos , Leucemia Mieloide Aguda/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Animales , Quinasa 6 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 6 Dependiente de la Ciclina/genética , Perfilación de la Expresión Génica , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Ratones , Proteínas de Complejo Poro Nuclear/genética , Proteínas de Fusión Oncogénica/genéticaRESUMEN
Over 80% of patients with myeloproliferative neoplasms (MPNs) harbor the acquired somatic JAK2 V617F mutation. JAK inhibition is not curative and fails to induce a persistent response in most patients, illustrating the need for the development of novel therapeutic approaches. We describe a critical role for CDK6 in MPN evolution. The absence of Cdk6 ameliorates clinical symptoms and prolongs survival. The CDK6 protein interferes with 3 hallmarks of disease: besides regulating malignant stem cell quiescence, it promotes nuclear factor κB (NF-κB) signaling and contributes to cytokine production while inhibiting apoptosis. The effects are not mirrored by palbociclib, showing that the functions of CDK6 in MPN pathogenesis are largely kinase independent. Our findings thus provide a rationale for targeting CDK6 in MPN.
Asunto(s)
Apoptosis , Quinasa 6 Dependiente de la Ciclina/farmacología , Janus Quinasa 2/genética , Mutación , Trastornos Mieloproliferativos/etiología , FN-kappa B/metabolismo , Humanos , Trastornos Mieloproliferativos/tratamiento farmacológico , Trastornos Mieloproliferativos/mortalidad , Trastornos Mieloproliferativos/patología , Neoplasias , Transducción de SeñalRESUMEN
Cyclin-dependent kinases 4 and 6 (CDK4/6) inhibitors are considered a breakthrough in cancer therapy. Currently approved for breast cancer treatment, CDK4/6 inhibitors are extensively tested in other cancer subtypes. Frequently observed side effects include hematological abnormalities such as reduced numbers of neutrophils, erythroid cells and platelets that are associated with anemia, bleeding and a higher risk of infections. In order to understand whether the adverse effects within the hematopoietic system are related to CDK4 or CDK6 we generated transgenic mice that lack either CDK4 or CDK6 in adult hematopoiesis. Anemia and perturbed erythroid differentiation are associated with the absence of CDK6 but did not manifest in CDK4- deficient mice. Total CDK6 knockout mice accumulate the most dormant fraction of hematopoietic stem cells due to an impaired exit of the quiescent state. We recapitulated this finding by deleting CDK6 in adult hematopoiesis. In addition, unlike total CDK6 knockout, all stem cell fractions were affected and increased in numbers. The deletion of CDK6 was also accompanied by neutropenia which is frequently seen in patients receiving CDK4/6 inhibitors. This was not the case in the absence of CDK4; CDK4 deficiency resulted in elevated numbers of myeloid progenitors without translating into numeric changes of differentiated myeloid cells. By using Cdk4fl/fl and Cdk6fl/fl mice we assign side effects of CDK4/6 inhibitors predominantly to the absence of CDK6. These mice represent a novel and powerful tool that will enable to study the distinct functions of CDK4 and CDK6 in a tissue-dependent manner.
Asunto(s)
Neoplasias de la Mama , Quinasa 4 Dependiente de la Ciclina , Quinasa 6 Dependiente de la Ciclina , Células Madre Hematopoyéticas , Animales , Quinasa 4 Dependiente de la Ciclina/genética , Quinasa 6 Dependiente de la Ciclina/genética , Femenino , Eliminación de Gen , Hematopoyesis/genética , Ratones , Ratones Noqueados , Ratones TransgénicosRESUMEN
Recurrent gain-of-function mutations in the transcription factors STAT5A and much more in STAT5B were found in hematopoietic malignancies with the highest proportion in mature T- and natural killer-cell neoplasms (peripheral T-cell lymphoma, PTCL). No targeted therapy exists for these heterogeneous and often aggressive diseases. Given the shortage of models for PTCL, we mimicked graded STAT5A or STAT5B activity by expressing hyperactive Stat5a or STAT5B variants at low or high levels in the hematopoietic system of transgenic mice. Only mice with high activity levels developed a lethal disease resembling human PTCL. Neoplasia displayed massive expansion of CD8+ T cells and destructive organ infiltration. T cells were cytokine-hypersensitive with activated memory CD8+ T-lymphocyte characteristics. Histopathology and mRNA expression profiles revealed close correlation with distinct subtypes of PTCL. Pronounced STAT5 expression and activity in samples from patients with different subsets underline the relevance of JAK/STAT as a therapeutic target. JAK inhibitors or a selective STAT5 SH2 domain inhibitor induced cell death and ruxolitinib blocked T-cell neoplasia in vivo We conclude that enhanced STAT5A or STAT5B action both drive PTCL development, defining both STAT5 molecules as targets for therapeutic intervention.
Asunto(s)
Leucemia , Linfoma de Células T Periférico , Animales , Linfocitos T CD8-positivos/metabolismo , Citocinas , Humanos , Linfoma de Células T Periférico/genética , Ratones , Factor de Transcripción STAT5/genética , Factor de Transcripción STAT5/metabolismo , Proteínas Supresoras de TumorRESUMEN
While significant progress has been made in the treatment of acute myeloid leukemia (AML), not all patients can be cured. Mutated in about 1/3 of de novo AML, the FLT3 receptor tyrosine kinase is an attractive target for drug development, activating mutations of the FLT3 map to the juxtamembrane domain (internal tandem duplications, ITD) or the tyrosine kinase domain (TKD), most frequently at codon D835. While small molecule tyrosine kinase inhibitors (TKI) effectively target ITD mutant forms, those on the TKD are not responsive. Moreover, FLT3 inhibition fails to induce a persistent response in patients due to mutational resistance. More potent compounds with broader inhibitory effects on multiple FLT3 mutations are highly desirable. We describe a critical role of CDK6 in the survival of FLT3⺠AML cells as palbociclib induced apoptosis not only in FLT3â»ITD⺠cells but also in FLT3â»D835Y⺠cells. Antineoplastic effects were also seen in primary patient-derived cells and in a xenograft model, where therapy effectively suppressed tumor formation in vivo at clinically relevant concentrations. In cells with FLT3â»ITD or -TKD mutations, the CDK6 protein not only affects cell cycle progression but also transcriptionally regulates oncogenic kinases mediating intrinsic drug resistance, including AURORA and AKT-a feature not shared by its homolog CDK4. While AKT and AURORA kinase inhibitors have significant therapeutic potential in AML, single agent activity has not been proven overly effective. We describe synergistic combination effects when applying these drugs together with palbociclib which could be readily translated to patients with AML bearing FLT3â»ITD or â»TKD mutations. Targeting synergistically acting vulnerabilities, with CDK6 being the common denominator, may represent a promising strategy to improve AML patient responses and to reduce the incidence of selection of resistance-inducing mutations.
Asunto(s)
Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/metabolismo , Piperazinas/farmacología , Piridinas/farmacología , Tirosina Quinasa 3 Similar a fms/genética , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Aurora Quinasas/genética , Aurora Quinasas/metabolismo , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Inmunoprecipitación de Cromatina , Quinasa 6 Dependiente de la Ciclina/genética , Quinasa 6 Dependiente de la Ciclina/metabolismo , Immunoblotting , Ratones , Mutación/genética , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Autosomal dominant polycystic kidney disease (ADPKD) leads to renal failure. The hallmark of ADPKD is increased epithelial proliferation, which has been proposed to be due to atypical signaling including abnormal JAK-STAT activity. However, the relative contribution of JAK-STAT family members in promoting proliferation in ADPKD is unknown. Here, we present siRNA JAK-STAT-focused screens discovering a previously unknown proliferative role for multiple JAK-STAT components (including STAT1, STAT2, STAT4, STAT5a, and STAT5b). Amongst these, we selected to study the growth hormone/growth hormone receptor/STAT5-axis because of its known role as a regulator of growth in nonrenal tissues. Loss of STAT5 function, facilitated by pharmacological inhibition or siRNAs, significantly reduced proliferation with an associated reduction in cyst growth in vitro. To study whether STAT5 is abnormally activated in vivo, we analyzed its expression using two independent mouse models of ADPKD. STAT5 was nuclear, thus activated, in renal epithelial cyst lining cells in both models. To test whether forced activation of STAT5 can modulate proliferation of renal cells in vivo, irrespective of the Pkd1 status, we overexpressed growth hormone. These mice showed increased STAT5 activity in renal epithelial cells, which correlated with de novo expression of cyclin D1, a STAT5 target gene. Chromatin immunoprecipitation experiments revealed that STAT5 transcriptionally activated cyclin D1 in a growth hormone-dependent fashion, thus providing a mechanism into how STAT5 enhances proliferation. Finally, we provide evidence of elevated serum growth hormone in Pkd1 mutant mice. Thus, the growth hormone/STAT5 signaling axis is a novel therapeutic target in ADPKD.
Asunto(s)
Proliferación Celular , Células Epiteliales/metabolismo , Riñón/metabolismo , Riñón Poliquístico Autosómico Dominante/metabolismo , Factor de Transcripción STAT5/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Proteínas Portadoras/metabolismo , Línea Celular , Núcleo Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Ciclina D1/genética , Ciclina D1/metabolismo , Modelos Animales de Enfermedad , Células Epiteliales/efectos de los fármacos , Células Epiteliales/patología , Genotipo , Hormona del Crecimiento/genética , Hormona del Crecimiento/metabolismo , Humanos , Quinasas Janus/antagonistas & inhibidores , Quinasas Janus/genética , Quinasas Janus/metabolismo , Riñón/efectos de los fármacos , Riñón/patología , Ratones Transgénicos , Fenotipo , Riñón Poliquístico Autosómico Dominante/tratamiento farmacológico , Riñón Poliquístico Autosómico Dominante/genética , Riñón Poliquístico Autosómico Dominante/patología , Inhibidores de Proteínas Quinasas/farmacología , Interferencia de ARN , Factor de Transcripción STAT5/antagonistas & inhibidores , Factor de Transcripción STAT5/genética , Transducción de Señal , Canales Catiónicos TRPP/genética , Canales Catiónicos TRPP/metabolismo , Factores de Tiempo , Transfección , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Proteínas Supresoras de Tumor/genéticaRESUMEN
T cell acute lymphoblastic leukemia (T-ALL) is an aggressive immature T cell cancer. Mutations in IL7R have been analyzed genetically, but downstream effector functions such as STAT5A and STAT5B hyperactivation are poorly understood. Here, we studied the most frequent and clinically challenging STAT5BN642H driver in T cell development and immature T cell cancer onset and compared it with STAT5A hyperactive variants in transgenic mice. Enhanced STAT5 activity caused disrupted T cell development and promoted an early T cell progenitor-ALL phenotype, with upregulation of genes involved in T cell receptor (TCR) signaling, even in absence of surface TCR. Importantly, TCR pathway genes were overexpressed in human T-ALL and mature T cell cancers and activation of TCR pathway kinases was STAT5 dependent. We confirmed STAT5 binding to these genes using ChIP-Seq analysis in human T-ALL cells, which were sensitive to pharmacologic inhibition by dual STAT3/5 degraders or ZAP70 tyrosine kinase blockers in vitro and in vivo. We provide genetic and biochemical proof that STAT5A and STAT5B hyperactivation can initiate T-ALL through TCR pathway hijacking and suggest similar mechanisms for other T cell cancers. Thus, STAT5 or TCR component blockade are targeted therapy options, particularly in patients with chemoresistant clones carrying STAT5BN642H.
Asunto(s)
Leucemia-Linfoma Linfoblástico de Células T Precursoras , Animales , Humanos , Ratones , Ratones Transgénicos , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Proteínas Tirosina Quinasas , Receptores de Antígenos de Linfocitos T/genética , Transducción de Señal , Factor de Transcripción STAT5/genéticaRESUMEN
Primary tenocytes rapidly undergo senescence and a phenotypic drift upon in vitro monolayer culture, which limits tendon research. The Ink4a/Arf locus encodes the proteins p16Ink4a/Arf and p14ARF (p19ARF in mice) that regulate cell cycle progression and senescence. We here established an immortalized cell line using tenocytes isolated from Ink4a/Arf deficient mice (Ink4a/Arf-/-). These cells were investigated at three distinct time points, at low (2-5), intermediate (14-17) and high (35-44) passages. Wild-type cells at low passage (2-5) served as controls. Ink4a/Arf-/- tenocytes at all stages were comparable to wild-type cells regarding morphology, expression of tenogeneic genes (collagen type 1, 3 and 5, Scleraxis, Tenomodulin and Tenascin-C), and surface markers (CD29, CD44 and CD105) and form 3D tendon-like structures. Importantly, Ink4a/Arf-/- tenocytes maintained their phenotypic features and proliferation potential in culture for more than 40 passages and also following freeze-thaw cycles. In contrast, wild-type tenocytes underwent senescence starting in passage 6. These data define Ink4a/Arf-/- tenocytes as novel tool for in vitro tendon research and as valuable in vitro alternative to animal experiments.
Asunto(s)
Inhibidor p16 de la Quinasa Dependiente de Ciclina , Tenocitos , Animales , Ratones , Tenocitos/metabolismo , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Proteína p14ARF Supresora de Tumor/genética , Tendones/metabolismo , Línea CelularRESUMEN
High levels of macrophage migration inhibitory factor (MIF) in patients with cancer are associated with poor prognosis. Its redox-dependent conformational isoform, termed oxidized MIF (oxMIF), is a promising tumor target due to its selective occurrence in tumor lesions and at inflammatory sites. A first-generation anti-oxMIF mAb, imalumab, was investigated in clinical trials in patients with advanced solid tumors, where it was well tolerated and showed signs of efficacy. However, imalumab has a short half-life in humans, increased aggregation propensity, and an unfavorable pharmacokinetic profile. Here, we aimed to optimize imalumab by improving its physicochemical characteristics and boosting its effector functions. Point mutations introduced into the variable regions reduced hydrophobicity and the antibodies' aggregation potential, and increased plasma half-life and tumor accumulation in vivo, while retaining affinity and specificity to oxMIF. The introduction of mutations into the Fc region known to increase antibody-dependent cellular cytotoxicity resulted in enhanced effector functions of the novel antibodies in vitro, whereas reduced cytokine release from human peripheral blood mononuclear cells in the absence of target antigen by the engineered anti-oxMIF mAb ON203 versus imalumab reveals a favorable in vitro safety profile. In vivo, ON203 mAb demonstrated superior efficacy over imalumab in both prophylactic and established prostate cancer (PC3) mouse xenograft models. In summary, our data highlight the potential of the second-generation anti-oxMIF mAb ON203 as a promising immunotherapy for patients with solid tumors, warranting clinical evaluation.
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Antineoplásicos , Factores Inhibidores de la Migración de Macrófagos , Neoplasias de la Próstata , Masculino , Ratones , Animales , Humanos , Factores Inhibidores de la Migración de Macrófagos/genética , Factores Inhibidores de la Migración de Macrófagos/química , Leucocitos Mononucleares , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/uso terapéutico , Neoplasias de la Próstata/tratamiento farmacológicoRESUMEN
Defining the role of genes in the genesis of congenital cardiovascular defects involves comparisons of the diameters of arteries measured in wild-type and genetically engineered mouse embryos. This study aims at evaluating the significance and reproducibility of measurements of the diameters of the great intrathoracic arteries of mouse embryos, as produced under routine conditions, by employing a recently suggested measuring method. Using high-resolution episcopic microscopy, we generated digital volume data of 60 mouse embryos (voxel size 1.07 × 1.07 × 2 µm(3)) of developmental stage 23 according to Theiler. We randomly split the 60 data sets into two groups of 30 and assigned each group to a diploma student. In addition, an experienced scientist received 12 randomly selected specimens of each group. Independently, the researchers created three-dimensional models of the intrathoracic arteries and identified comparable measurement positions along the ascending aorta, pulmonary trunk and descending aorta. At each position, they defined virtual resections cutting through the volume data perpendicular to the longitudinal axis of the artery. In the virtual resections, the researchers measured the perimeter of the lumen of the artery. The diameter was calculated from the perimeter. Then, we performed statistic comparisons of the diameters measured in micrometres and of the ratio of each measured diameter and the diameter of the ascending aorta. Comparisons of the ratios did not reveal statistically significant differences between the measurements created by the different scientists. We assume that the used measuring protocol is highly robust and produces reproducible and significant results under routine conditions.
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Arterias/anatomía & histología , Arterias/embriología , Embrión de Mamíferos/anatomía & histología , Embrión de Mamíferos/irrigación sanguínea , Microscopía/métodos , Estadística como Asunto , Animales , Aorta/anatomía & histología , Aorta/embriología , Femenino , Humanos , Personal de Laboratorio , Masculino , Ratones , Competencia Profesional , Reproducibilidad de los ResultadosRESUMEN
Studies of molecular mechanisms of hematopoiesis and leukemogenesis are hampered by the unavailability of progenitor cell lines that accurately mimic the situation in vivo. We now report a robust method to generate and maintain LSK (Lin-, Sca-1+, c-Kit+) cells, which closely resemble MPP1 cells. HPCLSKs reconstitute hematopoiesis in lethally irradiated recipient mice over >8 months. Upon transformation with different oncogenes including BCR/ABL, FLT3-ITD, or MLL-AF9, their leukemic counterparts maintain stem cell properties in vitro and recapitulate leukemia formation in vivo. The method to generate HPCLSKs can be applied to transgenic mice, and we illustrate it for CDK6-deficient animals. Upon BCR/ABLp210 transformation, HPCLSKsCdk6-/- induce disease with a significantly enhanced latency and reduced incidence, showing the importance of CDK6 in leukemia formation. Studies of the CDK6 transcriptome in murine HPCLSK and human BCR/ABL+ cells have verified that certain pathways depend on CDK6 and have uncovered a novel CDK6-dependent signature, suggesting a role for CDK6 in leukemic progenitor cell homing. Loss of CDK6 may thus lead to a defect in homing. The HPCLSK system represents a unique tool for combined in vitro and in vivo studies and enables the production of large quantities of genetically modifiable hematopoietic or leukemic stem/progenitor cells.
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Proteínas de Fusión bcr-abl , Células Madre Hematopoyéticas , Animales , Hematopoyesis , Ratones , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
IL-17-producing RORγt+ γδ T cells (γδT17 cells) are innate lymphocytes that participate in type 3 immune responses during infection and inflammation. Herein, we show that γδT17 cells rapidly proliferate within neonatal lymph nodes and gut, where, upon entry, they upregulate T-bet and coexpress IL-17, IL-22, and IFN-γ in a STAT3- and retinoic acid-dependent manner. Neonatal expansion was halted in mice conditionally deficient in STAT5, and its loss resulted in γδT17 cell depletion from all adult organs. Hyperactive STAT5 mutant mice showed that the STAT5A homolog had a dominant role over STAT5B in promoting γδT17 cell expansion and downregulating gut-associated T-bet. In contrast, STAT5B preferentially expanded IFN-γ-producing γδ populations, implying a previously unknown differential role of STAT5 gene products in lymphocyte lineage regulation. Importantly, mice lacking γδT17 cells as a result of STAT5 deficiency displayed a profound resistance to experimental autoimmune encephalomyelitis. Our data identify that the neonatal microenvironment in combination with STAT5 is critical for post-thymic γδT17 development and tissue-specific imprinting, which is essential for infection and autoimmunity.
Asunto(s)
Microambiente Celular/inmunología , Inmunidad Innata , Intestinos/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Factor de Transcripción STAT5/inmunología , Linfocitos T/inmunología , Animales , Animales Recién Nacidos , Microambiente Celular/genética , Citocinas/genética , Citocinas/inmunología , Intestinos/citología , Ratones , Ratones Transgénicos , Especificidad de Órganos/genética , Especificidad de Órganos/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/genética , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/inmunología , Factor de Transcripción STAT5/genética , Linfocitos T/citologíaRESUMEN
The transcription factor STAT3 is frequently activated in human solid and hematological malignancies and remains a challenging therapeutic target with no approved drugs to date. Here, we develop synthetic antibody mimetics, termed monobodies, to interfere with STAT3 signaling. These monobodies are highly selective for STAT3 and bind with nanomolar affinity to the N-terminal and coiled-coil domains. Interactome analysis detects no significant binding to other STATs or additional off-target proteins, confirming their exquisite specificity. Intracellular expression of monobodies fused to VHL, an E3 ubiquitin ligase substrate receptor, results in degradation of endogenous STAT3. The crystal structure of STAT3 in complex with monobody MS3-6 reveals bending of the coiled-coil domain, resulting in diminished DNA binding and nuclear translocation. MS3-6 expression strongly inhibits STAT3-dependent transcriptional activation and disrupts STAT3 interaction with the IL-22 receptor. Therefore, our study establishes innovative tools to interfere with STAT3 signaling by different molecular mechanisms.
Asunto(s)
Anticuerpos/metabolismo , Factor de Transcripción STAT3/metabolismo , Células A549 , Anticuerpos/genética , Western Blotting , Calorimetría , Cristalografía por Rayos X , Citometría de Flujo , Polarización de Fluorescencia , Técnica del Anticuerpo Fluorescente , Humanos , Espectrometría de Masas , Unión Proteica , Dominios Proteicos/inmunología , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/inmunología , Transducción de Señal/genética , Transducción de Señal/fisiología , Biología SintéticaRESUMEN
The transcription factors STAT5A and STAT5B have essential roles in survival and proliferation of hematopoietic cells-which have been considered largely redundant. Mutations of upstream kinases, copy number gains, or activating mutations in STAT5A, or more frequently in STAT5B, cause altered hematopoiesis and cancer. Interfering with their activity by pharmacological intervention is an up-and-coming therapeutic avenue. Precision medicine requests detailed knowledge of STAT5A's and STAT5B's individual functions. Recent evidence highlights the privileged role for STAT5B over STAT5A in normal and malignant hematopoiesis. Here, we provide an overview on their individual functions within the hematopoietic system.