RESUMEN
The long-term survival of plasma cells is entirely dependent on signals derived from their environment. These extrinsic factors presumably induce and sustain the expression of antiapoptotic proteins of the Bcl-2 family. It is uncertain whether there is specificity among Bcl-2 family members in the survival of plasma cells and whether their expression is linked to specific extrinsic factors. We found here that deletion of the gene encoding the antiapoptotic protein Mcl-1 in plasma cells resulted in rapid depletion of this population in vivo. Furthermore, we found that the receptor BCMA was needed to establish high expression of Mcl-1 in bone marrow but not spleen plasma cells and that establishing this survival pathway preceded the component of plasma cell differentiation that depends on the transcriptional repressor Blimp-1. Our results identify a critical role for Mcl-1 in the maintenance of plasma cells.
Asunto(s)
Antígeno de Maduración de Linfocitos B/metabolismo , Células Plasmáticas/fisiología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Factores de Transcripción/metabolismo , Animales , Médula Ósea/inmunología , Médula Ósea/metabolismo , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Diferenciación Celular , Supervivencia Celular , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Células Plasmáticas/citología , Factor 1 de Unión al Dominio 1 de Regulación Positiva , Proteínas Proto-Oncogénicas c-bcl-2/genética , Bazo/inmunologíaRESUMEN
Studies of gene-targeted mice identified the roles of the different pro-survival BCL-2 proteins during embryogenesis. However, little is known about the role(s) of these proteins in adults in response to cytotoxic stresses, such as treatment with anti-cancer agents. We investigated the role of BCL-XL in adult mice using a strategy where prior bone marrow transplantation allowed for loss of BCL-XL exclusively in non-hematopoietic tissues to prevent anemia caused by BCL-XL deficiency in erythroid cells. Unexpectedly, the combination of total body γ-irradiation (TBI) and genetic loss of Bcl-x caused secondary anemia resulting from chronic renal failure due to apoptosis of renal tubular epithelium with secondary obstructive nephropathy. These findings identify a critical protective role of BCL-XL in the adult kidney and inform on the use of BCL-XL inhibitors in combination with DNA damage-inducing drugs for cancer therapy. Encouragingly, the combination of DNA damage-inducing anti-cancer therapy plus a BCL-XL inhibitor could be tolerated in mice, at least when applied sequentially.
Asunto(s)
Anemia/prevención & control , Riñón/efectos de la radiación , Proteína bcl-X/metabolismo , Proteína bcl-X/farmacología , Animales , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/genética , Proteína 11 Similar a Bcl2/genética , Daño del ADN , Femenino , Rayos gamma , Neoplasias Hematológicas/patología , Inflamación , Riñón/metabolismo , Riñón/patología , Hígado/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Transcriptoma , Proteínas Supresoras de Tumor/genética , Proteína bcl-X/deficiencia , Proteína bcl-X/genéticaRESUMEN
Unlike clustered HOX genes, the role of nonclustered homeobox gene family members in hematopoiesis and leukemogenesis has not been extensively studied. Here we found that the hematopoietically expressed homeobox gene Hhex is overexpressed in acute myeloid leukemia (AML) and is essential for the initiation and propagation of MLL-ENL-induced AML but dispensable for normal myelopoiesis, indicating a specific requirement for Hhex for leukemic growth. Loss of Hhex leads to expression of the Cdkn2a-encoded tumor suppressors p16(INK4a) and p19(ARF), which are required for growth arrest and myeloid differentiation following Hhex deletion. Mechanistically, we show that Hhex binds to the Cdkn2a locus and directly interacts with the Polycomb-repressive complex 2 (PRC2) to enable H3K27me3-mediated epigenetic repression. Thus, Hhex is a potential therapeutic target that is specifically required for AML stem cells to repress tumor suppressor pathways and enable continued self-renewal.
Asunto(s)
Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Epigénesis Genética , Proteínas de Homeodominio/metabolismo , Leucemia Mieloide Aguda/fisiopatología , Complejo Represivo Polycomb 2/genética , Complejo Represivo Polycomb 2/metabolismo , Factores de Transcripción/metabolismo , Animales , Línea Celular Tumoral , Eliminación de Gen , Regulación Neoplásica de la Expresión Génica , Proteínas de Homeodominio/genética , Humanos , Leucemia Mieloide Aguda/genética , Ratones , Ratones Endogámicos C57BL , Unión Proteica , Factores de Transcripción/genéticaRESUMEN
The transcriptional regulator c-MYC is abnormally overexpressed in many human cancers. Evasion from apoptosis is critical for cancer development, particularly c-MYC-driven cancers. We explored which anti-apoptotic BCL-2 family member (expressed under endogenous regulation) is essential to sustain c-MYC-driven lymphoma growth to reveal which should be targeted for cancer therapy. Remarkably, inducible Cre-mediated deletion of even a single Mcl-1 allele substantially impaired the growth of c-MYC-driven mouse lymphomas. Mutations in p53 could diminish but not obviate the dependency of c-MYC-driven mouse lymphomas on MCL-1. Importantly, targeting of MCL-1 killed c-MYC-driven human Burkitt lymphoma cells, even those bearing mutations in p53. Given that loss of one allele of Mcl-1 is well tolerated in healthy tissues, our results suggest that therapeutic targeting of MCL-1 would be an attractive therapeutic strategy for MYC-driven cancers.
Asunto(s)
Linfoma/genética , Linfoma/terapia , Mutación , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteína p53 Supresora de Tumor/genética , Animales , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Linfoma/patología , Ratones , Ratones Endogámicos C57BL , Unión Proteica , Proteínas Proto-Oncogénicas c-myc/genéticaRESUMEN
Acute myeloid leukemia (AML) frequently relapses after initial treatment. Drug resistance in AML has been attributed to high levels of the anti-apoptotic Bcl-2 family members Bcl-x(L) and Mcl-1. Here we report that removal of Mcl-1, but not loss or pharmacological blockade of Bcl-x(L), Bcl-2, or Bcl-w, caused the death of transformed AML and could cure disease in AML-afflicted mice. Enforced expression of selective inhibitors of prosurvival Bcl-2 family members revealed that Mcl-1 is critical for survival of human AML cells. Thus, targeting of Mcl-1 or regulators of its expression may be a useful strategy for the treatment of AML.
Asunto(s)
Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Animales , Antineoplásicos Hormonales/farmacología , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Eliminación de Gen , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Ratones Endogámicos C57BL , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Tamoxifeno/farmacología , Células Tumorales CultivadasRESUMEN
The B-cell CLL/lymphoma 2 (Bcl2) relative Myeloid cell leukemia sequence 1 (Mcl1) is essential for cell survival during development and for tissue homeostasis throughout life. Unlike Bcl2, Mcl1 turns over rapidly, but the physiological significance of its turnover has been unclear. We have gained insight into the roles of Mcl1 turnover in vivo by analyzing mice harboring a modified allele of Mcl1 that serendipitously proved to encode an abnormally stabilized form of Mcl1 due to a 13-aa N-terminal extension. Although the mice developed normally and appeared unremarkable, the homozygous males unexpectedly proved infertile due to defective spermatogenesis, which was evoked by enhanced Mcl1 prosurvival activity. Under unstressed conditions, the modified Mcl1 is present at levels comparable to the native protein, but it is markedly stabilized in cells subjected to stresses, such as protein synthesis inhibition or UV irradiation. Strikingly, the modified Mcl1 allele could genetically complement the loss of Bcl2, because introduction of even a single allele significantly ameliorated the severe polycystic kidney disease and consequent runting caused by Bcl2 loss. Significantly, the development of c-MYC-induced acute myeloid leukemia was also accelerated in mice harboring that Mcl1 allele. Our collective findings reveal that, under certain circumstances, the N terminus of Mcl1 regulates its degradation; that some cell types require degradation of Mcl1 to induce apoptosis; and, most importantly, that rapid turnover of Mcl1 can serve as a tumor-suppressive mechanism.
Asunto(s)
Apoptosis , Transformación Celular Neoplásica/genética , Infertilidad Masculina/genética , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/fisiología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Alelos , Animales , Muerte Celular , Supervivencia Celular , Femenino , Fibroblastos/metabolismo , Citometría de Flujo , Regulación de la Expresión Génica , Células HEK293 , Humanos , Infertilidad Masculina/metabolismo , Leucemia Mieloide Aguda/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , Enfermedades Renales Poliquísticas/metabolismo , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Espermatogénesis , Testículo/patología , Factores de Tiempo , Rayos UltravioletaRESUMEN
When murine fetal liver cells were transduced with either of the human acute myeloid leukemia fusion oncogenes MLL-ENL or MLL-AF9 and then transplanted to irradiated recipient mice, myelomonocyte leukemias rapidly developed from the transplanted cells. Analysis of initial events following transduction showed that both oncogenes immediately induced a wide range of enhanced proliferative states, the most extreme of which could generate continuous lines of cells. Maturation defects accompanied the enhanced proliferative states. At all times, the transformed cells exhibited complete dependency on hematopoietic growth factors, particularly GM-CSF and IL-3. Myelomonocytic leukemic cells from primary or transplanted mice formed colonies in semisolid agar. The large majority were dependent on hematopoietic growth factors, but a low frequency of autonomous colonies was also detected. Unexpectedly, reculture of autonomous leukemic colonies generated large numbers of growth factor-dependent clonogenic progeny. Similarly, transplanted clonal autonomous leukemic cells produced leukemias containing a majority of factor-dependent cells. Conversely, recultures of factor-dependent colonies in vitro always produced small numbers of autonomous colonies among the dependent progeny. The reversible relationship between factor dependency and autonomy is surprising because autonomy would have been presumed to represent the final, irreversible, leukemic state.
Asunto(s)
Transformación Celular Neoplásica/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Interleucina-3/farmacología , Leucemia Mieloide/metabolismo , Proteína de la Leucemia Mieloide-Linfoide/biosíntesis , Proteínas de Fusión Oncogénica/biosíntesis , Animales , Línea Celular , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Humanos , Interleucina-3/metabolismo , Leucemia Mieloide/genética , Leucemia Mieloide/patología , Ratones , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteínas de Fusión Oncogénica/genéticaRESUMEN
Resistance to cell death is a hallmark of cancer and renders transformed cells resistant to multiple apoptotic triggers. The Bcl-2 family member, Mcl-1, is a key driver of cell survival in diverse cancers, including acute myeloid leukemia (AML). A screen for compounds that downregulate Mcl-1 identified the kinase inhibitor, PIK-75, which demonstrates marked proapoptotic activity against a panel of cytogenetically diverse primary human AML patient samples. We show that PIK-75 transiently blocks Cdk7/9, leading to transcriptional suppression of MCL-1, rapid loss of Mcl-1 protein, and alleviation of its inhibition of proapoptotic Bak. PIK-75 also targets the p110α isoform of PI3K, which leads to a loss of association between Bcl-xL and Bak. The simultaneous loss of Mcl-1 and Bcl-xL association with Bak leads to rapid apoptosis of AML cells. Concordantly, low Bak expression in AML confers resistance to PIK-75-mediated killing. On the other hand, the induction of apoptosis by PIK-75 did not require the expression of the BH3 proteins Bim, Bid, Bad, Noxa, or Puma. PIK-75 significantly reduced leukemia burden and increased the survival of mice engrafted with human AML without inducing overt toxicity. Future efforts to cotarget PI3K and Cdk9 with drugs such as PIK-75 in AML are warranted.
Asunto(s)
Quinasa 9 Dependiente de la Ciclina/antagonistas & inhibidores , Leucemia Mieloide Aguda/tratamiento farmacológico , Terapia Molecular Dirigida/métodos , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas c-bcl-2/genética , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Células Cultivadas , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Células HEK293 , Células HL-60 , Humanos , Hidrazonas/uso terapéutico , Leucemia Mieloide Aguda/patología , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Inhibidores de Proteínas Quinasas/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Sulfonamidas/uso terapéutico , Transcripción Genética/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The BH3-mimetic ABT-737 and an orally bioavailable compound of the same class, navitoclax (ABT-263), have shown promising antitumor efficacy in preclinical and early clinical studies. Although both drugs avidly bind Bcl-2, Bcl-x(L), and Bcl-w in vitro, we find that Bcl-2 is the critical target in vivo, suggesting that patients with tumors overexpressing Bcl-2 will probably benefit. In human non-Hodgkin lymphomas, high expression of Bcl-2 but not Bcl-x(L) predicted sensitivity to ABT-263. Moreover, we show that increasing Bcl-2 sensitized normal and transformed lymphoid cells to ABT-737 by elevating proapoptotic Bim. In striking contrast, increasing Bcl-x(L) or Bcl-w conferred robust resistance to ABT-737, despite also increasing Bim. Cell-based protein redistribution assays unexpectedly revealed that ABT-737 disrupts Bcl-2/Bim complexes more readily than Bcl-x(L)/Bim or Bcl-w/Bim complexes. These results have profound implications for how BH3-mimetics induce apoptosis and how the use of these compounds can be optimized for treating lymphoid malignancies.
Asunto(s)
Compuestos de Anilina/farmacología , Proteínas Reguladoras de la Apoptosis/antagonistas & inhibidores , Compuestos de Bifenilo/farmacología , Leucemia/tratamiento farmacológico , Linfoma/tratamiento farmacológico , Terapia Molecular Dirigida , Nitrofenoles/farmacología , Sulfonamidas/farmacología , Proteína bcl-X/antagonistas & inhibidores , Compuestos de Anilina/uso terapéutico , Animales , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína 11 Similar a Bcl2 , Compuestos de Bifenilo/uso terapéutico , Muerte Celular/efectos de los fármacos , Citoprotección/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Etopósido/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Leucemia/genética , Leucemia/patología , Linfoma/genética , Linfoma/patología , Proteínas de la Membrana/metabolismo , Ratones , Proteínas Mutantes/metabolismo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Nitrofenoles/uso terapéutico , Piperazinas/farmacología , Piperazinas/uso terapéutico , Unión Proteica/efectos de los fármacos , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Sulfonamidas/uso terapéutico , Proteína Destructora del Antagonista Homólogo bcl-2/metabolismo , Proteína X Asociada a bcl-2/metabolismo , Proteína bcl-X/metabolismoRESUMEN
BACKGROUND: Cellular barcoding is a recently developed biotechnology tool that enables the familial identification of progeny of individual cells in vivo. In immunology, it has been used to track the burst-sizes of multiple distinct responding T cells over several adaptive immune responses. In the study of hematopoiesis, it revealed fate heterogeneity amongst phenotypically identical multipotent cells. Most existing approaches rely on ex vivo viral transduction of cells with barcodes followed by adoptive transfer into an animal, which works well for some systems, but precludes barcoding cells in their native environment such as those inside solid tissues. RESULTS: With a view to overcoming this limitation, we propose a new design for a genetic barcoding construct based on the Cre Lox system that induces randomly created stable barcodes in cells in situ by exploiting inherent sequence distance constraints during site-specific recombination. We identify the cassette whose provably maximal code diversity is several orders of magnitude higher than what is attainable with previously considered Cre Lox barcoding approaches, exceeding the number of lymphocytes or hematopoietic progenitor cells in mice. CONCLUSIONS: Its high diversity and in situ applicability, make the proposed Cre Lox based tagging system suitable for whole tissue or even whole animal barcoding. Moreover, it can be built using established technology.
Asunto(s)
Ingeniería Genética/métodos , Integrasas/metabolismo , Recombinación Genética , Animales , Linfocitos T CD8-positivos/metabolismo , Variación Genética , Secuencias Invertidas Repetidas/genética , RatonesRESUMEN
Inhibition of the apoptosis pathway controlled by opposing members of the Bcl-2 protein family plays a central role in cancer development and resistance to therapy. To investigate how pro-apoptotic Bcl-2 homology domain 3 (BH3)-only proteins impact on acute myeloid leukemia (AML), we generated mixed lineage leukemia (MLL)-AF9 and MLL-ENL AMLs from BH3-only gene knockout mice. Disease development was not accelerated by loss of Bim, Puma, Noxa, Bmf, or combinations thereof; hence these BH3-only proteins are apparently ineffectual as tumor suppressors in this model. We tested the sensitivity of MLL-AF9 AMLs of each genotype in vitro to standard chemotherapeutic drugs and to the proteasome inhibitor bortezomib, with or without the BH3 mimetic ABT-737. Loss of Puma and/or Noxa increased resistance to cytarabine, daunorubicin and etoposide, while loss of Bim protected against cytarabine and loss of Bmf had no impact. ABT-737 increased sensitivity to the genotoxic drugs but was not dependent on any BH3-only protein tested. The AML lines were very sensitive to bortezomib and loss of Noxa conveyed significant resistance. In vivo, several MLL-AF9 AMLs responded well to daunorubicin and this response was highly dependent on Puma and Noxa but not Bim. Combination therapy with ABT-737 provided little added benefit at the daunorubicin dose trialed. Bortezomib also extended survival of AML-bearing mice, albeit less than daunorubicin. In summary, our genetic studies reveal the importance of Puma and Noxa for the action of genotoxics currently used to treat MLL-driven AML and suggest that, while addition of ABT-737-like BH3 mimetics might enhance their efficacy, new Noxa-like BH3 mimetics targeting Mcl-1 might have greater potential.
Asunto(s)
Carcinogénesis/patología , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteínas de Fusión Oncogénica/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Compuestos de Bifenilo/farmacología , Compuestos de Bifenilo/uso terapéutico , Carcinogénesis/efectos de los fármacos , Carcinogénesis/genética , Daunorrubicina/farmacología , Daunorrubicina/uso terapéutico , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Técnicas de Inactivación de Genes , Leucemia Mieloide Aguda/patología , Ratones Endogámicos C57BL , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Nitrofenoles/farmacología , Nitrofenoles/uso terapéutico , Proteínas de Fusión Oncogénica/metabolismo , Piperazinas/farmacología , Piperazinas/uso terapéutico , Sulfonamidas/farmacología , Sulfonamidas/uso terapéuticoRESUMEN
The Suppressors of Cytokine Signalling (SOCS) proteins are negative regulators of cytokine signalling required to prevent excess cellular responses. SOCS1 and SOCS3 are essential to prevent inflammatory disease, SOCS1 by attenuating responses to IFNγ and gamma-common (γc) cytokines, and SOCS3 via regulation of G-CSF and IL-6 signalling. SOCS1 and SOCS3 show significant sequence homology and are the only SOCS proteins to possess a KIR domain. The possibility of overlapping or redundant functions was investigated in inflammatory disease via generation of mice lacking both SOCS1 and SOCS3 in hematopoietic cells. Loss of SOCS3 significantly accelerated the pathology and inflammatory disease characteristic of SOCS1 deficiency. We propose a model in which SOCS1 and SOCS3 operate independently to control specific cytokine responses and together modulate the proliferation and activation of lymphoid and myeloid cells to prevent rapid inflammatory disease.
Asunto(s)
Células de la Médula Ósea/metabolismo , Inflamación/metabolismo , Proteína 1 Supresora de la Señalización de Citocinas/metabolismo , Proteína 3 Supresora de la Señalización de Citocinas/metabolismo , Animales , Linfocitos T CD8-positivos/citología , Factor Estimulante de Colonias de Granulocitos/biosíntesis , Inflamación/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína 1 Supresora de la Señalización de Citocinas/genética , Proteína 3 Supresora de la Señalización de Citocinas/genéticaRESUMEN
Human pathogenic Legionella replicate in alveolar macrophages and cause a potentially lethal form of pneumonia known as Legionnaires' disease(1). Here, we have identified a host-directed therapeutic approach to eliminate intracellular Legionella infections. We demonstrate that the genetic deletion, or pharmacological inhibition, of the host cell pro-survival protein BCL-XL induces intrinsic apoptosis of macrophages infected with virulent Legionella strains, thereby abrogating Legionella replication. BCL-XL is essential for the survival of Legionella-infected macrophages due to bacterial inhibition of host-cell protein synthesis, resulting in reduced levels of the short-lived, related BCL-2 pro-survival family member, MCL-1. Consequently, a single dose of a BCL-XL-targeted BH3-mimetic therapy, or myeloid cell-restricted deletion of BCL-XL, limits Legionella replication and prevents lethal lung infections in mice. These results indicate that repurposing BH3-mimetic compounds, originally developed to induce cancer cell apoptosis, may have efficacy in treating Legionnaires' and other diseases caused by intracellular microbes.
Asunto(s)
Apoptosis , Legionella/crecimiento & desarrollo , Macrófagos Alveolares/microbiología , Macrófagos Alveolares/fisiología , Proteína bcl-X/antagonistas & inhibidores , Animales , Modelos Animales de Enfermedad , Eliminación de Gen , Legionelosis/prevención & control , Ratones , Proteína bcl-X/genéticaRESUMEN
Pro-survival BCL-2 family members protect cells from programmed cell death that can be induced by multiple internal or external cues. Within the haematopoietic lineages, the BCL-2 family members BCL-2, BCL-XL and MCL-1 are known to support cell survival but the individual and overlapping roles of these pro-survival BCL-2 proteins for the persistence of individual leukocyte subsets in vivo has not yet been determined. By combining inducible knockout mouse models with the BH3-mimetic compound ABT-737, which inhibits BCL-2, BCL-XL and BCL-W, we found that dependency on MCL-1, BCL-XL or BCL-2 expression changes during B-cell development. We show that BCL-XL expression promotes survival of immature B cells, expression of BCL-2 is important for survival of mature B cells and long-lived plasma cells (PC), and expression of MCL-1 is important for survival throughout B-cell development. These data were confirmed with novel highly specific BH3-mimetic compounds that target either BCL-2, BCL-XL or MCL-1. In addition, we observed that combined inhibition of these pro-survival proteins acts in concert to delete specific B-cell subsets. Reduced expression of MCL-1 further sensitized immature as well as transitional B cells and splenic PC to loss of BCL-XL expression. More markedly, loss of MCL-1 greatly sensitizes PC populations to BCL-2 inhibition using ABT-737, even though the total wild-type PC pool in the spleen is not significantly affected by this drug and the bone marrow (BM) PC population only slightly. Combined loss or inhibition of MCL-1 and BCL-2 reduced the numbers of established PC >100-fold within days. Our data suggest that combination treatment targeting these pro-survival proteins could be advantageous for treatment of antibody-mediated autoimmune diseases and B-cell malignancies.
Asunto(s)
Linfocitos B/metabolismo , Subgrupos Linfocitarios/metabolismo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Proteína bcl-X/metabolismo , Animales , Antígeno de Maduración de Linfocitos B/metabolismo , Linfocitos B/efectos de los fármacos , Compuestos de Bifenilo/farmacología , Subgrupos Linfocitarios/efectos de los fármacos , Ratones Endogámicos C57BL , Modelos Biológicos , Nitrofenoles/farmacología , Piperazinas/farmacología , Células Plasmáticas/efectos de los fármacos , Células Plasmáticas/metabolismo , Sulfonamidas/farmacologíaRESUMEN
Resistance to chemotherapy is a major problem in cancer treatment, and it is frequently associated with failure of tumor cells to undergo apoptosis. Birinapant, a clinical SMAC mimetic, had been designed to mimic the interaction between inhibitor of apoptosis proteins (IAPs) and SMAC/Diablo, thereby relieving IAP-mediated caspase inhibition and promoting apoptosis of cancer cells. We show that acute myeloid leukemia (AML) cells are sensitive to birinapant-induced death and that the clinical caspase inhibitor emricasan/IDN-6556 augments, rather than prevents, killing by birinapant. Deletion of caspase-8 sensitized AML to birinapant, whereas combined loss of caspase-8 and the necroptosis effector MLKL (mixed lineage kinase domain-like) prevented birinapant/IDN-6556-induced death, showing that inhibition of caspase-8 sensitizes AML cells to birinapant-induced necroptosis. However, loss of MLKL alone did not prevent a caspase-dependent birinapant/IDN-6556-induced death, implying that AML will be less likely to acquire resistance to this drug combination. A therapeutic breakthrough in AML has eluded researchers for decades. Demonstrated antileukemic efficacy and safety of the birinapant/emricasan combination in vivo suggest that induction of necroptosis warrants clinical investigation as a therapeutic opportunity in AML.
Asunto(s)
Caspasa 8/metabolismo , Inhibidores de Caspasas/farmacología , Dipéptidos/farmacología , Indoles/farmacología , Ácidos Pentanoicos/farmacología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Resistencia a Antineoplásicos/efectos de los fármacos , Sinergismo Farmacológico , Humanos , Péptidos y Proteínas de Señalización Intracelular , Leucemia Mieloide Aguda/metabolismo , Necrosis/metabolismo , Células Tumorales CultivadasRESUMEN
Birinapant is a smac-mimetic (SM) in clinical trials for treating cancer. SM antagonize inhibitor of apoptosis (IAP) proteins and simultaneously induce tumor necrosis factor (TNF) secretion to render cancers sensitive to TNF-induced killing. To enhance SM efficacy, we screened kinase inhibitors for their ability to increase TNF production of SM-treated cells. We showed that p38 inhibitors increased TNF induced by SM. Unexpectedly, even though p38 is required for Toll-like receptors to induce TNF, loss of p38 or its downstream kinase MK2 increased induction of TNF by SM. Hence, we show that the p38/MK2 axis can inhibit or promote TNF production, depending on the stimulus. Importantly, clinical p38 inhibitors overcame resistance of primary acute myeloid leukemia to birinapant.
Asunto(s)
Antineoplásicos/uso terapéutico , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/fisiología , Leucemia/tratamiento farmacológico , Proteínas Mitocondriales/fisiología , Imitación Molecular , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Animales , Antineoplásicos/farmacología , Proteínas Reguladoras de la Apoptosis , Humanos , Ratones , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
The cytokine IL-15 is required for natural killer (NK) cell homeostasis; however, the intrinsic mechanism governing this requirement remains unexplored. Here we identify the absolute requirement for myeloid cell leukaemia sequence-1 (Mcl1) in the sustained survival of NK cells in vivo. Mcl1 is highly expressed in NK cells and regulated by IL-15 in a dose-dependent manner via STAT5 phosphorylation and subsequent binding to the 3'-UTR of Mcl1. Specific deletion of Mcl1 in NK cells results in the absolute loss of NK cells from all tissues owing to a failure to antagonize pro-apoptotic proteins in the outer mitochondrial membrane. This NK lymphopenia results in mice succumbing to multiorgan melanoma metastases, being permissive to allogeneic transplantation and being resistant to toxic shock following polymicrobial sepsis challenge. These results clearly demonstrate a non-redundant pathway linking IL-15 to Mcl1 in the maintenance of NK cells and innate immune responses in vivo.
Asunto(s)
Eliminación de Gen , Enfermedades del Sistema Inmune/genética , Inmunidad Innata/genética , Células Asesinas Naturales/metabolismo , Células Asesinas Naturales/patología , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Animales , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Citocinas/farmacología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Femenino , Enfermedades del Sistema Inmune/fisiopatología , Inmunidad Innata/fisiología , Interleucina-15/farmacología , Interleucina-15/fisiología , Células Asesinas Naturales/efectos de los fármacos , Linfopenia/genética , Linfopenia/patología , Linfopenia/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Factor de Transcripción STAT5/fisiología , Transducción de Señal/fisiologíaRESUMEN
Deregulated expression of Hox genes such as HoxA9 is associated with development of myeloproliferative disorders and leukemia and indicates a poor prognosis. To investigate the molecular mechanisms by which HoxA9 promotes immortalization of hematopoietic cells, we generated growth factor dependent myeloid cells in which HoxA9 expression is regulated by administration of 4-hydroxy-tamoxifen. Maintenance of HoxA9 overexpression is required for continued cell survival and proliferation, even in the presence of growth factors. We show for the first time that maintenance of Bcl-2 expression is critical for HoxA9-dependent immortalization and influences the latency of HoxA9-dependent leukemia. Hematopoietic cells lacking Bcl-2 were not immortalized by HoxA9 in vitro. Furthermore, deletion of Bcl-2 delayed the onset and reduced the severity of HoxA9/Meis1 and MLL-AF9 leukemias. This is the first description of a molecular link between HoxA9 and the regulation of Bcl-2 family members in acute myeloid leukemia.