RESUMEN
ABSTRACT: Hematopoietic stem cells (HSCs) are characterized by the ability to self-renew and to replenish the hematopoietic system. The cell-cycle kinase cyclin-dependent kinase 6 (CDK6) regulates transcription, whereby it has both kinase-dependent and kinase-independent functions. Herein, we describe the complex role of CDK6, balancing quiescence, proliferation, self-renewal, and differentiation in activated HSCs. Mouse HSCs expressing kinase-inactivated CDK6 show enhanced long-term repopulation and homing, whereas HSCs lacking CDK6 have impaired functionality. The transcriptomes of basal and serially transplanted HSCs expressing kinase-inactivated CDK6 exhibit an expression pattern dominated by HSC quiescence and self-renewal, supporting a concept, in which myc-associated zinc finger protein (MAZ) and nuclear transcription factor Y subunit alpha (NFY-A) are critical CDK6 interactors. Pharmacologic kinase inhibition with a clinically used CDK4/6 inhibitor in murine and human HSCs validated our findings and resulted in increased repopulation capability and enhanced stemness. Our findings highlight a kinase-independent role of CDK6 in long-term HSC functionality. CDK6 kinase inhibition represents a possible strategy to improve HSC fitness.
Asunto(s)
Quinasa 6 Dependiente de la Ciclina , Células Madre Hematopoyéticas , Quinasa 6 Dependiente de la Ciclina/metabolismo , Quinasa 6 Dependiente de la Ciclina/genética , Animales , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/citología , Ratones , Humanos , Células Madre Adultas/metabolismo , Células Madre Adultas/citología , Proliferación Celular , Diferenciación Celular , Ratones Endogámicos C57BL , Trasplante de Células Madre Hematopoyéticas , Autorrenovación de las Células/efectos de los fármacosRESUMEN
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.
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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.
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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
Demonstrations of both pro-apoptotic and pro-survival abilities of Fas (TNFRSF6/CD95/APO-1) have led to a shift from the exclusive "Fas apoptosis" to "Fas multisignals" paradigm and the acceptance that Fas-related therapies face a major challenge, as it remains unclear what determines the mode of Fas signaling. Through protein evolution analysis, which reveals unconventional substitutions of Fas tyrosine during divergent evolution, evolution-guided tyrosine-phosphorylated Fas proxy, and site-specific phosphorylation detection, we show that the Fas signaling outcome is determined by the tyrosine phosphorylation status of its death domain. The phosphorylation dominantly turns off the Fas-mediated apoptotic signal, while turning on the pro-survival signal. We show that while phosphorylations at Y232 and Y291 share some common functions, their contributions to Fas signaling differ at several levels. The findings that Fas tyrosine phosphorylation is regulated by Src family kinases (SFKs) and the phosphatase SHP-1 and that Y291 phosphorylation primes clathrin-dependent Fas endocytosis, which contributes to Fas pro-survival signaling, reveals for the first time the mechanistic link between SFK/SHP-1-dependent Fas tyrosine phosphorylation, internalization route, and signaling choice. We also demonstrate that levels of phosphorylated Y232 and Y291 differ among human cancer types and differentially respond to anticancer therapy, suggesting context-dependent involvement of Fas phosphorylation in cancer. This report provides a new insight into the control of TNF receptor multisignaling by receptor phosphorylation and its implication in cancer biology, which brings us a step closer to overcoming the challenge in handling Fas signaling in treatments of cancer as well as other pathologies such as autoimmune and degenerative diseases.
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Evolución Molecular , Neoplasias/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 6/metabolismo , Receptor fas/metabolismo , Familia-src Quinasas/metabolismo , Secuencia de Aminoácidos , Apoptosis , Endocitosis , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fosforilación , Estructura Terciaria de ProteínaRESUMEN
The mammalian skin is the largest organ of the body and its outermost layer, the epidermis, undergoes dynamic lifetime renewal through the activity of somatic stem cell populations. The EGFR-Ras-Raf pathway has a well-described role in skin development and tumor formation. While research mainly focuses on its role in cutaneous tumor initiation and maintenance, much less is known about Ras signaling in the epidermal stem cells, which are the main targets of skin carcinogenesis. In this review, we briefly discuss the properties of the epidermal stem cells and review the role of EGFR-Ras-Raf signaling in keratinocyte stem cells during homeostatic and pathological conditions.
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Epidermis/fisiología , Receptores ErbB/genética , Folículo Piloso/fisiología , Neoplasias Cutáneas/fisiopatología , Células Madre/fisiología , Quinasas raf/genética , Proteínas ras/genética , Animales , Epidermis/metabolismo , Receptores ErbB/metabolismo , Folículo Piloso/metabolismo , Humanos , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/metabolismo , Células Madre/metabolismo , Quinasas raf/metabolismo , Proteínas ras/metabolismoRESUMEN
Cellular fibronectin (cFN) variants harboring extra FN type 3 repeats, namely extra domains B and A, are major constituents of the extracellular matrix around newly forming blood vessels during development and angiogenesis. Their expression is induced by angiogenic stimuli and their assembly into fibrillar arrays is driven by cell-generated tension at α5ß1 integrin-based adhesions. Here, we examined the role and functional redundancy of cFN variants in cultured endothelial cells by isoform-selective RNA interference. We show that FN fibrillogenesis is a cell-autonomous process whereby basally directed secretion and assembly of cellular FN are tightly coupled events that play an important role not only in signaling at cell-matrix adhesions but also at cell-cell contacts. Silencing of cFN variants differentially affects integrin usage, cell spreading, motility and capillary morphogenesis in vitro. cFN-deficient cells undergo a switch from α5ß1- to αvß3-based adhesion, accompanied by a Src-regulated disruption of adherens junctions. These studies identify a crucial role for autocrine FN in subendothelial matrix assembly and junctional integrity that provides spatially and temporally restricted control of endothelial plasticity during angiogenic blood vessel remodeling.
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Comunicación Celular/fisiología , Células Endoteliales/fisiología , Fibronectinas/fisiología , Adhesión Celular/fisiología , Ciclo Celular/fisiología , Uniones Célula-Matriz/metabolismo , Células Endoteliales/citología , Células Endoteliales/metabolismo , Matriz Extracelular/metabolismo , Matriz Extracelular/fisiología , Fibronectinas/metabolismo , Humanos , Integrinas/metabolismo , Transducción de Señal , TransfecciónRESUMEN
Hematopoietic stem cells (HSCs) are rare, self-renewing cells that perch on top of the hematopoietic tree. The HSCs ensure the constant supply of mature blood cells in a tightly regulated process producing peripheral blood cells. Intense efforts are ongoing to optimize HSC engraftment as therapeutic strategy to treat patients suffering from hematopoietic diseases. Preclinical research paves the way by developing methods to maintain, manipulate and expand HSCs ex vivo to understand their regulation and molecular make-up. The generation of a sufficient number of transplantable HSCs is the Holy Grail for clinical therapy. Leukemia stem cells (LSCs) are characterized by their acquired stem cell characteristics and are responsible for disease initiation, progression, and relapse. We summarize efforts, that have been undertaken to increase the number of long-term (LT)-HSCs and to prevent differentiation towards committed progenitors in ex vivo culture. We provide an overview and compare methods currently available to isolate, maintain and enrich HSC subsets, progenitors and LSCs and discuss their individual advantages and drawbacks.
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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
The oncogenic protein Bcr-Abl has two major isoforms, p190Bcr-Abl and p210Bcr-Abl. While p210Bcr-Abl is the hallmark of chronic myeloid leukemia (CML), p190Bcr-Abl occurs in the majority of Philadelphia-positive acute lymphoblastic leukemia (Ph + ALL) patients. In CML, p190Bcr-Abl occurs in a minority of patients associating with distinct hematological features and inferior outcomes, yet the pathogenic role of p190Bcr-Abl and potential targeting therapies are largely uncharacterized. We employed next generation sequencing, phospho-proteomic profiling, and drug sensitivity testing to characterize p190Bcr-Abl in CML and hematopoietic progenitor cell line models (Ba/f3 and HPC-LSK). p190Bcr-Abl CML patients demonstrated poor response to imatinib and frequent mutations in epigenetic modifiers genes. In contrast with p210Bcr-Abl, p190Bcr-Abl exhibited specific transcriptional upregulation of interferon, interleukin-1 receptor, and P53 signaling pathways, associated with hyperphosphorylation of relevant signaling molecules including JAK1/STAT1 and PAK1 in addition to Src hyperphosphorylation. Comparable to p190Bcr-Abl CML patients, p190Bcr-Abl cell lines demonstrated similar transcriptional and phospho-signaling signatures. With the drug sensitivity screening we identified targeted drugs with specific activity in p190Bcr-Abl cell lines including IAP-, PAK1-, and Src inhibitors and glucocorticoids. Our results provide novel insights into the mechanisms underlying the distinct features of p190Bcr-Abl CML and promising therapeutic targets for this high-risk patient group.
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Proteínas de Fusión bcr-abl/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Transducción de Señal/genética , Anciano , Anciano de 80 o más Años , Animales , Línea Celular Tumoral , Femenino , Perfilación de la Expresión Génica/métodos , Glucocorticoides/genética , Humanos , Masculino , Ratones , Persona de Mediana Edad , Oncogenes/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteómica/métodos , Transcripción Genética/genética , Regulación hacia Arriba/genéticaRESUMEN
The endothelium functions as a semipermeable barrier regulating fluid homeostasis, nutrient, and gas supply to the tissue. Endothelial permeability is increased in several pathological conditions including inflammation and tumors; despite its clinical relevance, however, there are no specific therapies preventing vascular leakage. Here, we show that endothelial cell-restricted ablation of BRAF, a kinase frequently activated in cancer, prevents vascular leaking as well metastatic spread. BRAF regulates endothelial permeability by promoting the cytoskeletal rearrangements necessary for the remodeling of VE-Cadherin-containing endothelial cell-cell junctions and the formation of intercellular gaps. BRAF kinase activity and the ability to form complexes with RAS/RAP1 and dimers with its paralog RAF1 are required for proper permeability control, achieved mechanistically by modulating the interaction between RAF1 and the RHO effector ROKα. Thus, RAF dimerization impinges on RHO pathways to regulate cytoskeletal rearrangements, junctional plasticity, and endothelial permeability. The data advocate the development of RAF dimerization inhibitors, which would combine tumor cell autonomous effect with stabilization of the vasculature and antimetastatic spread.
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Antígenos CD/genética , Cadherinas/genética , Citoesqueleto/genética , Proteínas Proto-Oncogénicas B-raf/genética , Quinasas Asociadas a rho/genética , Animales , Permeabilidad Capilar/genética , Citoesqueleto/metabolismo , Dimerización , Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Humanos , Uniones Intercelulares/genética , Ratones , Fosforilación/genética , Factor Rho/genética , Transducción de Señal , Proteínas de Unión al GTP rap1/genéticaRESUMEN
Tumor formation is a multistep process during which cells acquire genetic and epigenetic changes until they reach a fully transformed state. We show that CDK6 contributes to tumor formation by regulating transcriptional responses in a stage-specific manner. In early stages, the CDK6 kinase induces a complex transcriptional program to block p53 in hematopoietic cells. Cells lacking CDK6 kinase function are required to mutate TP53 (encoding p53) to achieve a fully transformed immortalized state. CDK6 binds to the promoters of genes including the p53 antagonists Prmt5, Ppm1d, and Mdm4 The findings are relevant to human patients: Tumors with low levels of CDK6 have mutations in TP53 significantly more often than expected.Significance: CDK6 acts at the interface of p53 and RB by driving cell-cycle progression and antagonizing stress responses. While sensitizing cells to p53-induced cell death, specific inhibition of CDK6 kinase activity may provoke the outgrowth of p53-mutant clones from premalignant cells. Cancer Discov; 8(7); 884-97. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 781.
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Carcinogénesis , Quinasa 6 Dependiente de la Ciclina/metabolismo , Mutación , Neoplasias/metabolismo , Proteína p53 Supresora de Tumor/genética , Animales , Línea Celular Tumoral , Transformación Celular Neoplásica , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Neoplasias/genéticaRESUMEN
The Raf/Mek/Erk signaling pathway, activated downstream of Ras primarily to promote proliferation, represents the best studied of the evolutionary conserved MAPK cascades. The investigation of the pathway has continued unabated since its discovery roughly 30 years ago. In the last decade, however, the identification of unexpected in vivo functions of pathway components, as well as the discovery of Raf mutations in human cancer, the ensuing quest for inhibitors, and the efforts to understand their mechanism of action, have boosted interest tremendously. From this large body of work, protein-protein interaction has emerged as a recurrent, crucial theme. This review focuses on the role of protein complexes in the regulation of the Raf/Mek/Erk pathway and in its cross-talk with other signaling cascades. Mapping these interactions and finding a way of exploiting them for therapeutic purposes is one of the challenges of future molecule-targeted therapy.
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Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Sistema de Señalización de MAP Quinasas , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Quinasas raf/metabolismo , Animales , Humanos , Unión ProteicaRESUMEN
RAF inhibitors achieve unprecedented but mainly transient clinical responses in patients with melanoma whose tumors harbor an activating BRAF mutation. One notable side-effect of RAF inhibitors is the stimulation of cutaneous skin tumors, arising in about 30% of patients receiving these drugs, which are thought to develop as a result of inhibitor-induced activation of wild-type Raf in occult precursor skin lesions. This effect raises the possibility that less manageable tumors might also arise in other epithelial tissues. Here we provide preclinical evidence supporting this disquieting hypothesis by showing that the RAF inhibitors PLX-4032 (vemurafenib) and GDC-0879 precipitate the development of cell-autonomous, Ras-driven tumors in skin and gastric epithelia. The magnitude of the effects correlated with the inhibitors' relative abilities to induce ERK activation. Epidermis-restricted ablation of either B-Raf or C-Raf prevented PLX-4032-induced ERK activation and tumorigenesis. In contrast, GDC-0879 induced ERK activation and tumorigenesis in B-Raf-deficient epidermis, whereas C-Raf ablation blocked GDC-0879-induced tumorigenesis (despite strong ERK activation) by preventing Rokα-mediated keratinocyte dedifferentiation. Thus, inhibitor-induced ERK activation did not require a specific Raf kinase. ERK activation was necessary, but not sufficient for Ras + Raf inhibitor-induced tumorigenesis, whereas C-Raf downregulation of Rokα was essential even in the face of sustained ERK signaling to prevent differentiation and promote tumorigenesis. Taken together, our findings suggest that combination therapies targeting ERK-dependent and -independent functions of Raf may be more efficient but also safer for cancer treatment.
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Carcinogénesis/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/fisiología , Melanoma/inducido químicamente , Inhibidores de Proteínas Quinasas/efectos adversos , Neoplasias Cutáneas/inducido químicamente , Quinasas raf/antagonistas & inhibidores , Quinasas raf/fisiología , Animales , Células COS , Chlorocebus aethiops , Humanos , Indenos/farmacología , Indoles/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Ratones Transgénicos , Pirazoles/farmacología , Sulfonamidas/farmacología , Células Tumorales Cultivadas , VemurafenibRESUMEN
The activation of cysteine-aspartic proteases or caspases and the dynamic arrangement of cytoskeletal components are crucial during apoptosis. Here we describe the fate of Fas downstream of the FasL-induced internalization step, including formation of caspase-dependent SDS-stable Fas complexes, which is mediated by cytoskeleton integrity. We show, in particular, that following FasL treatment, the Fas lower aggregate complex can be co-immunoprecipitated with tubulin and an active form of caspase-8 and that this interaction contributes to the propagation of FasL-induced cell death. The importance of cytoskeletal components during FasL-induced apoptosis is highlighted by our detection of a pool of microtubule-associated Fas complexes.