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In this issue of Molecular Cell, Cao et al. (2021) report that AML cells are specifically addicted to an IRF8-MEF2D gene expression network. Furthermore, they identify a chromatin reader, ZMYND8, as the upstream regulator of the IRF8-MEF2D program whose activity is critical for AML cell survival.
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Leucemia Mieloide Aguda , Proteínas Supressoras de Tumor , Cromatina , Humanos , Fatores Reguladores de Interferon/genética , Leucemia Mieloide Aguda/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
Tet enzymes (Tet1/2/3) oxidize 5-methylcytosine to promote DNA demethylation and partner with chromatin modifiers to regulate gene expression. Tet1 is highly expressed in embryonic stem cells (ESCs), but its enzymatic and non-enzymatic roles in gene regulation are not dissected. We have generated Tet1 catalytically inactive (Tet1m/m) and knockout (Tet1-/-) ESCs and mice to study these functions. Loss of Tet1, but not loss of its catalytic activity, caused aberrant upregulation of bivalent (H3K4me3+; H3K27me3+) developmental genes, leading to defects in differentiation. Wild-type and catalytic-mutant Tet1 occupied similar genomic loci which overlapped with H3K27 tri-methyltransferase PRC2 and the deacetylase complex Sin3a at promoters of bivalent genes and with the helicase Chd4 at active genes. Loss of Tet1, but not loss of its catalytic activity, impaired enrichment of PRC2 and Sin3a at bivalent promoters leading to reduced H3K27 trimethylation and deacetylation, respectively, in absence of any changes in DNA methylation. Tet1-/-, but not Tet1m/m, embryos expressed higher levels of Gata6 and were developmentally delayed. Thus, the critical functions of Tet1 in ESCs and early development are mediated through its non-catalytic roles in regulating H3K27 modifications to silence developmental genes, and are more important than its catalytic functions in DNA demethylation.
Assuntos
Proteínas de Ligação a DNA , Dioxigenases , Células-Tronco Embrionárias , Proteínas Proto-Oncogênicas , Animais , Diferenciação Celular/genética , DNA/metabolismo , Metilação de DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Dioxigenases/genética , Dioxigenases/metabolismo , Células-Tronco Embrionárias/metabolismo , Camundongos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismoRESUMO
This Defining Moments essay contributes to health communication scholarship regarding the role of religion/spirituality as a coping strategy during severe illness and the realities of providing social support in blended families. It also invites readers to reconsider the capacities and constraints of utilizing technology-mediated communication to provide support within close familial relationships. Finally, this narrative acknowledges the messiness of providing support in the evolving realities of family life and the complexity of "being there" for people we love when we cannot physically be present.
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OBJECTIVES: To investigate key factors that may contribute to the variability of rituximab-mediated peripheral and renal B cell depletion (BCD) in SLE. METHODS: We analysed: (i) CD19+ B cell counts in patients with SLE before and 1, 2, 3 and 6 months after treatment with rituximab, comparing them with RA patients; (ii) the presence of B cells in renal biopsies after rituximab therapy; (iii) whether the duration of BCD correlated with patient demographics and B cell expression of CD20 and FcγRIIb; and (iv) the effect of B cell activation factor (BAFF) on the efficiency of rituximab and obinutuzumab at inducing BCD in whole blood assays, in vitro. RESULTS: In SLE (n = 71), the duration of BCD was shorter compared with RA (n = 27). B cells were detectable in renal biopsy samples (n = 6) after treatment with rituximab in all patients with poor response while peripheral blood B cells remained low or undetectable in the same patients. There were no significant relationships between peripheral BCD and patient age, disease duration, serum C3 levels or the level of expression of B cell surface proteins CD20 and FcγRIIb. Obinutuzumab was more efficient than rituximab at inducing BCD in whole blood assays, regardless of excess BAFF. CONCLUSIONS: BCD in SLE is less efficient than in RA. Renal B cell presence following rituximab treatment was associated with poor outcomes. No significant relationships between any measured B cell related, clinical or laboratory parameters and the efficiency of BCD by rituximab was found. Obinutuzumab was superior to rituximab at inducing BCD.
Assuntos
Lúpus Eritematoso Sistêmico , Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Monoclonais Humanizados/uso terapêutico , Antígenos CD20 , Linfócitos B , Humanos , Rituximab/farmacologia , Rituximab/uso terapêuticoRESUMO
Pu.1 is an ETS family transcription factor (TF) that plays critical roles in erythroid progenitors by promoting proliferation and blocking terminal differentiation. However, the mechanisms controlling expression and down-regulation of Pu.1 during early erythropoiesis have not been defined. In this study, we identify the actions of Runx1 and Pu.1 itself at the Pu.1 gene Upstream Regulatory Element (URE) as major regulators of Pu.1 expression in Burst-Forming Unit erythrocytes (BFUe). During early erythropoiesis, Runx1 and Pu.1 levels decline, and chromatin accessibility at the URE is lost. Ectopic expression of Runx1 or Pu.1, both of which bind the URE, prevents Pu.1 down-regulation and blocks terminal erythroid differentiation, resulting in extensive ex vivo proliferation and immortalization of erythroid progenitors. Ectopic expression of Runx1 in BFUe lacking a URE fails to block terminal erythroid differentiation. Thus, Runx1, acting at the URE, and Pu.1 itself directly regulate Pu.1 levels in erythroid cells, and loss of both factors is critical for Pu.1 down-regulation during terminal differentiation. The molecular mechanism of URE inactivation in erythroid cells through loss of TF binding represents a distinct pattern of Pu.1 regulation from those described in other hematopoietic cell types such as T cells which down-regulate Pu.1 through active repression. The importance of down-regulation of Runx1 and Pu.1 in erythropoiesis is further supported by genome-wide analyses showing that their DNA-binding motifs are highly overrepresented in regions that lose chromatin accessibility during early erythroid development.
Assuntos
Diferenciação Celular/genética , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Células Precursoras Eritroides/citologia , Células Precursoras Eritroides/metabolismo , Regulação da Expressão Gênica , Proteínas Proto-Oncogênicas/genética , Transativadores/genética , Animais , Cromatina/genética , Cromatina/metabolismo , Eritropoese/genética , Camundongos , Células RAW 264.7 , Elementos de Resposta , Transcrição GênicaRESUMO
High levels of expression of wild-type Flt3 characterize many hematopoietic proliferative diseases and neoplasms, providing a potential therapeutic target. Using the c-Cbl RING finger mutant mouse as a model of a myeloproliferative disease (MPD) driven by wild-type Flt3, in the present study, we show that treatment with the Flt3 kinase inhibitor AC220 blocks MPD development by targeting Flt3(+) multipotent progenitors (MPPs). We found that daily administration of AC220 caused a marked reduction in Flt3 expression, induction of quiescence, and a significant loss of MPPs within 4 days. Unexpectedly, a robust Flt3 ligand-associated proliferative recovery response soon followed, preventing further loss of MPPs. However, continued AC220 treatment limited MPP recovery and maintained reduced, steady-state levels of cycling MPPs that express low levels of Flt3. Therefore, a finely tuned balance between the opposing forces of AC220 and Flt3 ligand production was established; whereas the Flt3 ligand blunted the inhibitory effects of AC220, the disease was held in remission for as long as therapy was continued. The net effect is a potent therapy indicating that patients with c-Cbl mutations, or those with similarly enhanced Flt3 signaling, may respond well to AC220 even after the induction of high levels of Flt3 ligand.
Assuntos
Benzotiazóis/farmacologia , Transtornos Mieloproliferativos/genética , Transtornos Mieloproliferativos/metabolismo , Compostos de Fenilureia/farmacologia , Transdução de Sinais/efeitos dos fármacos , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidores , Tirosina Quinase 3 Semelhante a fms/metabolismo , Animais , Benzotiazóis/administração & dosagem , Ciclo Celular/efeitos dos fármacos , Proliferação de Células , Modelos Animais de Doenças , Feminino , Humanos , Contagem de Leucócitos , Leucócitos/efeitos dos fármacos , Fígado/patologia , Pulmão/patologia , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Células-Tronco Multipotentes/efeitos dos fármacos , Células-Tronco Multipotentes/metabolismo , Mutação , Células Mieloides/patologia , Transtornos Mieloproliferativos/tratamento farmacológico , Compostos de Fenilureia/administração & dosagem , Proteínas Proto-Oncogênicas c-cbl/genética , Esplenomegalia/tratamento farmacológicoRESUMO
Phosphorylation is a key post-translational modification that is utilised in many biological processes for the rapid and reversible regulation of protein localisation and activity. Polo-like kinase 1 (PLK-1) is essential for both mitotic and meiotic cell divisions, with key functions being conserved in eukaryotes. The roles and regulation of PLK-1 during mitosis have been well characterised. However, the discrete roles and regulation of PLK-1 during meiosis have remained obscure. Here, we used Caenorhabditis elegans oocytes to show that PLK-1 plays distinct roles in meiotic spindle assembly and/or stability, chromosome alignment and segregation, and polar body extrusion during meiosis I. Furthermore, by a combination of live imaging and biochemical analysis we identified the chromosomal recruitment mechanisms of PLK-1 during C. elegans oocyte meiosis. The spindle assembly checkpoint kinase BUB-1 directly recruits PLK-1 to the kinetochore and midbivalent while the chromosome arm population of PLK-1 depends on a direct interaction with the centromeric-associated protein CENP-CHCP-4. We found that perturbing both BUB-1 and CENP-CHCP-4 recruitment of PLK-1 leads to severe meiotic defects, resulting in highly aneuploid oocytes. Overall, our results shed light on the roles played by PLK-1 during oocyte meiosis and provide a mechanistic understanding of PLK-1 targeting to meiotic chromosomes.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Segregação de Cromossomos , Cinetocoros , Meiose , Oócitos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Fuso Acromático/metabolismo , Quinase 1 Polo-LikeRESUMO
The master transcriptional regulator PU.1/Spi-1 engages DNA sites with affinities spanning multiple orders of magnitude. To elucidate this remarkable plasticity, we have characterized 22 high-resolution co-crystallographic PU.1/DNA complexes across the addressable affinity range in myeloid gene transactivation. Over a purine-rich core (such as 5'-GGAA-3') flanked by variable sequences, affinity is negotiated by direct readout on the 5' flank via a critical glutamine (Q226) sidechain and by indirect readout on the 3' flank by sequence-dependent helical flexibility. Direct readout by Q226 dynamically specifies PU.1's characteristic preference for purines and explains the pathogenic mutation Q226E in Waldenström macroglobulinemia. The structures also reveal how disruption of Q226 mediates strand-specific inhibition by DNA methylation and the recognition of non-canonical sites, including the authentic binding sequence at the CD11b promoter. A re-synthesis of phylogenetic and structural data on the ETS family, considering the centrality of Q226 in PU.1, unifies the model of DNA selection by ETS proteins.
Assuntos
DNA , Transativadores , Filogenia , Sítios de Ligação , Transativadores/metabolismo , DNA/metabolismoRESUMO
The principal aim of the study was to determine the influence of influenza A virus infection on capsaicin-induced relaxation responses in mouse isolated tracheal segments and clarify the underlying mechanisms. Anesthetized mice were intranasally inoculated with influenza A/PR-8/34 virus (VIRUS) or vehicle (SHAM), and 4 days later tracheal segments were harvested for isometric tension recording and biochemical and histologic analyses. Capsaicin induced dose-dependent relaxation responses in carbachol-contracted SHAM trachea (e.g., 10 µM capsaicin produced 66 ± 4% relaxation; n = 11), which were significantly inhibited by capsazepine [transient receptor potential vanilloid type 1 (TRPV1) antagonist], (2S,3S)-3-{[3,5-bis(trifluoromethyl)phenyl]methoxy}-2-phenylpiperidine hydrochloride (L-733,060) [neurokinin 1 (NK1) receptor antagonist], indomethacin [cyclooxygenase (COX) inhibitor], and the combination of 6-isopropoxy-9-oxoxanthene-2-carboxylic acid (AH6809) and 7-[5α-([1S,1α(Z)-biphenyl]-4-ylmethoxy)-2ß-(4-morpholinyl)-3-oxocyclopentyl]-4-heptenoic acid, calcium salt, hydrate (AH23848) [E-prostanoid (EP)2 and EP4 receptor antagonists, respectively], indicating that capsaicin-induced relaxation involved the TRPV1-mediated release of substance P (SP), activation of epithelial NK1 receptors, and production of COX products capable of activating relaxant EP2/EP4 receptors. Consistent with this postulate, capsaicin-induced relaxation was associated with the significant release of SP and prostaglandin E2 (PGE2) from mouse tracheal segments. As expected, influenza A virus infection was associated with widespread disruption of the tracheal epithelium. Tracheal segments from VIRUS mice responded weakly to capsaicin (7 ± 3% relaxation) and were 25-fold less responsive to SP than tracheas from SHAM mice. In contrast, relaxation responses to exogenous PGE2 and the ß-adrenoceptor agonist isoprenaline were not inhibited in VIRUS trachea. Virus infection was associated with impaired capsaicin-induced release of PGE2, but the release of SP was not affected. In summary, influenza A virus infection profoundly inhibits capsaicin- and SP-induced relaxation responses, most likely by inhibiting the production of PGE2.
Assuntos
Capsaicina/farmacologia , Vírus da Influenza A , Relaxamento Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Infecções por Orthomyxoviridae/fisiopatologia , Traqueia/fisiopatologia , Animais , Compostos de Bifenilo/farmacologia , Líquido da Lavagem Broncoalveolar/citologia , Capsaicina/análogos & derivados , Carbacol/farmacologia , Inibidores de Ciclo-Oxigenase/farmacologia , Dinoprostona/metabolismo , Dinoprostona/farmacologia , Relação Dose-Resposta a Droga , Indometacina/farmacologia , Inflamação/patologia , Inflamação/virologia , Isoproterenol/farmacologia , Leucócitos/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Modelos Biológicos , Contração Muscular/efeitos dos fármacos , Infecções por Orthomyxoviridae/metabolismo , Infecções por Orthomyxoviridae/patologia , Piperidinas/farmacologia , Antagonistas de Prostaglandina/farmacologia , Mucosa Respiratória/patologia , Mucosa Respiratória/virologia , Organismos Livres de Patógenos Específicos , Substância P/metabolismo , Substância P/farmacologia , Canais de Cátion TRPV/antagonistas & inibidores , Traqueia/efeitos dos fármacos , Traqueia/metabolismo , Traqueia/patologia , Traqueia/virologia , Xantonas/farmacologiaRESUMO
MDMX is overexpressed in the vast majority of patients with acute myeloid leukemia (AML). We report that MDMX overexpression increases preleukemic stem cell (pre-LSC) number and competitive advantage. Utilizing five newly generated murine models, we found that MDMX overexpression triggers progression of multiple chronic/asymptomatic preleukemic conditions to overt AML. Transcriptomic and proteomic studies revealed that MDMX overexpression exerts this function, unexpectedly, through activation of Wnt/ß-Catenin signaling in pre-LSCs. Mechanistically, MDMX binds CK1α and leads to accumulation of ß-Catenin in a p53-independent manner. Wnt/ß-Catenin inhibitors reverse MDMX-induced pre-LSC properties, and synergize with MDMX-p53 inhibitors. Wnt/ß-Catenin signaling correlates with MDMX expression in patients with preleukemic myelodysplastic syndromes and is associated with increased risk of progression to AML. Our work identifies MDMX overexpression as a pervasive preleukemic-to-AML transition mechanism in different genetically driven disease subtypes, and reveals Wnt/ß-Catenin as a non-canonical MDMX-driven pathway with therapeutic potential for progression prevention and cancer interception.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Leucemia Mieloide Aguda/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , beta Catenina/metabolismo , Animais , Linhagem Celular Tumoral , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Camundongos , Síndromes Mielodisplásicas/tratamento farmacológico , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/metabolismo , Proteômica/métodos , Via de Sinalização Wnt/genética , Via de Sinalização Wnt/fisiologiaRESUMO
The role of the kinetochore during meiotic chromosome segregation in C. elegans oocytes has been a matter of controversy. Danlasky et al. (2020. J. Cell. Biol.https://doi.org/10.1083/jcb.202005179) show that kinetochore proteins KNL-1 and KNL-3 are required for early stages of anaphase during female meiosis, suggesting a new kinetochore-based model of chromosome segregation.
Assuntos
Proteínas de Caenorhabditis elegans , Segregação de Cromossomos , Anáfase , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Feminino , Cinetocoros , Meiose , Proteínas Associadas aos Microtúbulos/genética , OócitosRESUMO
Protein Phosphatase 2A (PP2A) is a heterotrimer composed of scaffolding (A), catalytic (C), and regulatory (B) subunits. PP2A complexes with B56 subunits are targeted by Shugoshin and BUBR1 to protect centromeric cohesion and stabilise kinetochore-microtubule attachments in yeast and mouse meiosis. In Caenorhabditis elegans, the closest BUBR1 orthologue lacks the B56-interaction domain and Shugoshin is not required for meiotic segregation. Therefore, the role of PP2A in C. elegans female meiosis is unknown. We report that PP2A is essential for meiotic spindle assembly and chromosome dynamics during C. elegans female meiosis. BUB-1 is the main chromosome-targeting factor for B56 subunits during prometaphase I. BUB-1 recruits PP2A:B56 to the chromosomes via a newly identified LxxIxE motif in a phosphorylation-dependent manner, and this recruitment is important for proper chromosome congression. Our results highlight a novel mechanism for B56 recruitment, essential for recruiting a pool of PP2A involved in chromosome congression during meiosis I.
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Proteínas de Caenorhabditis elegans/fisiologia , Caenorhabditis elegans/fisiologia , Cromossomos/fisiologia , Meiose/fisiologia , Oócitos/fisiologia , Proteína Fosfatase 2/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Segregação de Cromossomos , Oócitos/metabolismo , Proteína Fosfatase 2/metabolismo , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
Mosaic evolution refers to the pattern whereby different organismal traits exhibit differential rates of evolution typically due to reduced levels of trait covariation through deep time (i.e., modularity). These differences in rates can be attributed to variation in responses to selective pressures between individual traits. Differential responses to selective pressures also have the potential to facilitate functional specialization, allowing certain traits to track environmental stimuli more closely than others. The teleost skull is a multifunctional structure comprising a complex network of bones and thus an excellent system for which to study mosaic evolution. Here we construct an ultrametric phylogeny for a clade of Neotropical electric fishes (Apteronotidae: Gymnotiformes) and use three-dimensional geometric morphometrics to investigate patterns of mosaic evolution in the skull and jaws. We find strong support for a developmental, three-module hypothesis that consists of the face, braincase, and mandible, and we find that the mandible has evolved four times faster than its neighboring modules. We hypothesize that the functional specialization of the mandible in this group of fishes has allowed it to outpace the face and braincase and evolve in a more decoupled manner. We also hypothesize that this pattern of mosaicism may be widespread across other clades of teleost fishes.
Assuntos
Evolução Biológica , Gimnotiformes/anatomia & histologia , Crânio/anatomia & histologia , Animais , FilogeniaRESUMO
The surface molecule interleukin-1 receptor accessory protein (IL1RAP) is consistently overexpressed across multiple genetic subtypes of acute myeloid leukemia (AML) and other myeloid malignancies, including at the stem cell level, and is emerging as a novel therapeutic target. However, the cell-intrinsic functions of IL1RAP in AML cells are largely unknown. Here, we show that targeting of IL1RAP via RNA interference, genetic deletion, or antibodies inhibits AML pathogenesis in vitro and in vivo, without perturbing healthy hematopoietic function or viability. Furthermore, we found that the role of IL1RAP is not restricted to the IL-1 receptor pathway, but that IL1RAP physically interacts with and mediates signaling and pro-proliferative effects through FLT3 and c-KIT, two receptor tyrosine kinases with known key roles in AML pathogenesis. Our study provides a new mechanistic basis for the efficacy of IL1RAP targeting in AML and reveals a novel role for this protein in the pathogenesis of the disease.
Assuntos
Carcinogênese/metabolismo , Carcinogênese/patologia , Proteína Acessória do Receptor de Interleucina-1/metabolismo , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Transdução de Sinais , Adulto , Idoso , Animais , Anticorpos/farmacologia , Apoptose/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Feminino , Deleção de Genes , Hematopoese/efeitos dos fármacos , Humanos , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Pessoa de Meia-Idade , Modelos Biológicos , Ligação Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-kit/metabolismo , Fator de Células-Tronco/metabolismoRESUMO
Myelosuppression is one of the most severe and limiting side effects of chemotherapy. Our recent work outlines a strategy to prevent chemotherapy-induced myelosuppression by administering a priming dose of the FMS-Like Tyrosine kinase 3 (FLT3) inhibitor quizartinib. Furthermore, by administering sequential quizartinib primed injections of fluorouracil (5-FU), we demonstrated a novel and effective strategy to eliminate disease in two mouse models of quizartinib resistant acute myeloid leukemia (AML).
RESUMO
We describe an approach to inhibit chemotherapy-induced myelosuppression. We found that short-term exposure of mice to the FLT3 inhibitor quizartinib induced the transient quiescence of multipotent progenitors (MPPs). This property of quizartinib conferred marked protection to MPPs in mice receiving fluorouracil or gemcitabine. The protection resulted in the rapid recovery of bone marrow and blood cellularity, thus preventing otherwise lethal myelosuppression. A treatment strategy involving quizartinib priming that protected wild-type bone marrow progenitors, but not leukemic cells, from fluorouracil provided a more effective treatment than conventional induction therapy in mouse models of acute myeloid leukemia. This strategy has the potential to be extended for use in other cancers where FLT3 inhibition does not adversely affect the effectiveness of chemotherapy. Thus, the addition of quizartinib to cancer treatment regimens could markedly improve cancer patient survival and quality of life.
Assuntos
Benzotiazóis/uso terapêutico , Compostos de Fenilureia/uso terapêutico , Tirosina Quinase 3 Semelhante a fms/metabolismo , Animais , Antineoplásicos/uso terapêutico , Proliferação de Células/efeitos dos fármacos , Fluoruracila/uso terapêutico , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Qualidade de Vida , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidoresRESUMO
Dasatinib is an orally available broad-spectrum tyrosine kinase inhibitor that is widely used to treat chronic myeloid leukemia. It is also in clinical trials for the treatment of other malignancies, including solid tumors. Despite its wide use, little is known of its effects on normal hematopoietic stem and progenitor cells. Here, we study wild-type mice dosed with dasatinib and find that it causes the transient induction of proliferation of quiescent hematopoietic stem cells (HSCs). This finding was unexpected given the ability of dasatinib to inhibit c-Kit signaling and promote cell cycle arrest in many cell types. The transient induction of HSC proliferation in dasatinib-dosed mice coincided with a marked induction in the expression of Sca-1 and phospho-S6. Also evident at this time was a rapid but transient loss of lineage-committed hematopoietic progenitors that express high levels of c-Kit and the induction of stem cell factor in the serum. These findings suggest that activation of quiescent HSCs is part of a rapid rescue response that restores hematopoietic progenitors to pretreatment levels. This restoration coincides with HSCs returning to quiescence, and the expression of Sca-1 and phospho-S6 reverting to pre-treatment levels, even though dasatinib dosing is maintained. These data suggest that equilibrium is reached between the opposing forces of dasatinib and hematopoietic growth factors. The transient induction of HSC proliferation provided a window of opportunity whereby these cells became sensitive to killing by the cytotoxic drug 5-fluorouracil.
Assuntos
Linhagem da Célula/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Dasatinibe/farmacologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Animais , Antígenos Ly/metabolismo , Antimetabólitos/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Citometria de Fluxo , Fluoruracila/farmacologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Mesilato de Imatinib/farmacologia , Masculino , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-kit/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Células-Tronco/sangue , Fator de Células-Tronco/metabolismo , Fatores de TempoRESUMO
Dasatinib inhibits B-cell receptor-Abelson murine leukemia viral oncogene homologue 1, Src, and other tyrosine kinases. Few studies have addressed the impact of dasatinib on normal blood cells, especially in vivo. Here we show that dasatinib leads to a reduced number of human CD19+ peripheral B cells owing to a strong induction of apoptosis. In contrast, no similar effect on T-cell viability was observed. However, dasatinib induced a comparable broad inhibition of the early events of B- and T-cell receptor signaling. Furthermore, dasatinib was shown to be a more pronounced inhibitor of both basal and B-cell receptor-induced activity of Bruton's tyrosine kinase and PLCγ2 compared with the more specific Bruton's tyrosine kinase inhibitor ibrutinib. Human progenitor B cells from the pre-B stage were sensitive to dasatinib. In an in vivo murine model, dasatinib reduced B-lineage cells in the bone marrow with a marked effect on the pre-B subpopulation. Dasatinib led to a reduced spleen size, with a loss of large immature transitional immunoglobulin M(+)/immunoglobulin D(-) B cells and a reduction in germinal center B cells. Dasatinib caused a marked loss of thymocytes without affecting myeloid lineage cells or hematopoietic progenitors. This study reveals important side effects of dasatinib with specific loss of activated B and thymocyte populations, which may have an impact during long-term treatment.
Assuntos
Apoptose/efeitos dos fármacos , Linfócitos B/efeitos dos fármacos , Pirimidinas/farmacologia , Tiazóis/farmacologia , Adenina/análogos & derivados , Tirosina Quinase da Agamaglobulinemia , Animais , Antígenos CD19/metabolismo , Linfócitos B/metabolismo , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Dasatinibe , Citometria de Fluxo , Humanos , Masculino , Camundongos Endogâmicos C57BL , Fosfolipase C gama/antagonistas & inibidores , Fosfolipase C gama/metabolismo , Piperidinas , Células Precursoras de Linfócitos B/efeitos dos fármacos , Células Precursoras de Linfócitos B/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Pirazóis/farmacologia , Receptores de Antígenos de Linfócitos B/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais/efeitos dos fármacos , Baço/citologia , Baço/efeitos dos fármacos , Baço/metabolismo , Fatores de TempoRESUMO
Mutations in the Fms-like tyrosine kinase 3 (FLT3) receptor tyrosine kinase (RTK) occur frequently in acute myeloid leukemia (AML), with the most common involving internal tandem duplication (ITD) within the juxtamembrane domain. Fms-like tyrosine kinase 3-ITD mutations result in a mislocalized and constitutively activated receptor, which aberrantly phosphorylates signal transducer and activator of transcription 5 (STAT5) and upregulates the expression of its target genes. c-Cbl is an E3 ubiquitin ligase that negatively regulates RTKs, including FLT3, but whether it can downregulate mislocalized FLT3-ITD remains to be resolved. To help clarify this, we combined a FLT3-ITD mutation with a loss-of-function mutation in the RING finger domain of c-Cbl that abolishes its E3 ligase activity. Mice transplanted with hematopoietic stem cells expressing both mutations rapidly develop myeloid leukemia, indicating strong cooperation between the two. Although the c-Cbl mutation was shown to cause hyperactivation of another RTK, c-Kit, it had no effect on enhancing FLT3-ITD protein levels or STAT5 activation. This indicates that c-Cbl does not downregulate FLT3-ITD and that the leukemia is driven by independent pathways involving FLT3-ITD's activation of STAT5 and mutant c-Cbl's activation of other RTKs, such as c-Kit. This study highlights the importance of c-Cbl's negative regulation of wild-type RTKs in suppressing FLT3-ITD-driven myeloid leukemia.