RESUMEN
R-2-hydroxyglutarate (R-2HG), produced at high levels by mutant isocitrate dehydrogenase 1/2 (IDH1/2) enzymes, was reported as an oncometabolite. We show here that R-2HG also exerts a broad anti-leukemic activity in vitro and in vivo by inhibiting leukemia cell proliferation/viability and by promoting cell-cycle arrest and apoptosis. Mechanistically, R-2HG inhibits fat mass and obesity-associated protein (FTO) activity, thereby increasing global N6-methyladenosine (m6A) RNA modification in R-2HG-sensitive leukemia cells, which in turn decreases the stability of MYC/CEBPA transcripts, leading to the suppression of relevant pathways. Ectopically expressed mutant IDH1 and S-2HG recapitulate the effects of R-2HG. High levels of FTO sensitize leukemic cells to R-2HG, whereas hyperactivation of MYC signaling confers resistance that can be reversed by the inhibition of MYC signaling. R-2HG also displays anti-tumor activity in glioma. Collectively, while R-2HG accumulated in IDH1/2 mutant cancers contributes to cancer initiation, our work demonstrates anti-tumor effects of 2HG in inhibiting proliferation/survival of FTO-high cancer cells via targeting FTO/m6A/MYC/CEBPA signaling.
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Antineoplásicos/farmacología , Neoplasias Encefálicas/tratamiento farmacológico , Glioma/tratamiento farmacológico , Glutaratos/farmacología , Leucemia/tratamiento farmacológico , Transducción de Señal/efectos de los fármacos , Adenosina/análogos & derivados , Adenosina/metabolismo , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/metabolismo , Animales , Antineoplásicos/uso terapéutico , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Línea Celular Tumoral , Glutaratos/uso terapéutico , Células HEK293 , Humanos , Células Jurkat , Ratones , Proteínas Proto-Oncogénicas c-myc/metabolismo , Procesamiento Postranscripcional del ARNRESUMEN
Dysregulation of innate immune signaling is a hallmark of hematologic malignancies. Recent therapeutic efforts to subvert aberrant innate immune signaling in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) have focused on the kinase IRAK4. IRAK4 inhibitors have achieved promising, though moderate, responses in preclinical studies and clinical trials for MDS and AML. The reasons underlying the limited responses to IRAK4 inhibitors remain unknown. In this study, we reveal that inhibiting IRAK4 in leukemic cells elicits functional complementation and compensation by its paralog, IRAK1. Using genetic approaches, we demonstrate that cotargeting IRAK1 and IRAK4 is required to suppress leukemic stem/progenitor cell (LSPC) function and induce differentiation in cell lines and patient-derived cells. Although IRAK1 and IRAK4 are presumed to function primarily downstream of the proximal adapter MyD88, we found that complementary and compensatory IRAK1 and IRAK4 dependencies in MDS/AML occur via noncanonical MyD88-independent pathways. Genomic and proteomic analyses revealed that IRAK1 and IRAK4 preserve the undifferentiated state of MDS/AML LSPCs by coordinating a network of pathways, including ones that converge on the polycomb repressive complex 2 complex and JAK-STAT signaling. To translate these findings, we implemented a structure-based design of a potent and selective dual IRAK1 and IRAK4 inhibitor KME-2780. MDS/AML cell lines and patient-derived samples showed significant suppression of LSPCs in xenograft and in vitro studies when treated with KME-2780 as compared with selective IRAK4 inhibitors. Our results provide a mechanistic basis and rationale for cotargeting IRAK1 and IRAK4 for the treatment of cancers, including MDS/AML.
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Leucemia Mieloide Aguda , Síndromes Mielodisplásicos , Humanos , Quinasas Asociadas a Receptores de Interleucina-1/genética , Quinasas Asociadas a Receptores de Interleucina-1/metabolismo , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/metabolismo , Proteómica , Transducción de Señal , Síndromes Mielodisplásicos/tratamiento farmacológico , Síndromes Mielodisplásicos/genética , Leucemia Mieloide Aguda/genéticaRESUMEN
The fusion gene MLL/AF4 defines a high-risk subtype of pro-B acute lymphoblastic leukemia. Relapse can be associated with a lineage switch from acute lymphoblastic to acute myeloid leukemia, resulting in poor clinical outcomes caused by resistance to chemotherapies and immunotherapies. In this study, the myeloid relapses shared oncogene fusion breakpoints with their matched lymphoid presentations and originated from various differentiation stages from immature progenitors through to committed B-cell precursors. Lineage switching is linked to substantial changes in chromatin accessibility and rewiring of transcriptional programs, including alternative splicing. These findings indicate that the execution and maintenance of lymphoid lineage differentiation is impaired. The relapsed myeloid phenotype is recurrently associated with the altered expression, splicing, or mutation of chromatin modifiers, including CHD4 coding for the ATPase/helicase of the nucleosome remodelling and deacetylation complex. Perturbation of CHD4 alone or in combination with other mutated epigenetic modifiers induces myeloid gene expression in MLL/AF4+ cell models, indicating that lineage switching in MLL/AF4 leukemia is driven and maintained by disrupted epigenetic regulation.
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Proteína de la Leucemia Mieloide-Linfoide , Leucemia-Linfoma Linfoblástico de Células Precursoras , Humanos , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Epigénesis Genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Genes Reguladores , CromatinaRESUMEN
Inhibitors of anti-apoptotic BCL-2 family proteins in combination with chemotherapy and hypomethylating agents (HMAs) are promising therapeutic approaches in acute myeloid leukemia (AML) and high-risk myelodysplastic syndromes (MDS). Alvocidib, a cyclin-dependent kinase 9 (CDK9) inhibitor and indirect transcriptional repressor of the anti-apoptotic factor MCL-1, has previously shown clinical activity in AML. Availability of biomarkers for response to the alvocidib + 5- AZA could also extend the rationale of this treatment concept to high-risk MDS. In this study, we performed a comprehensive in vitro assessment of alvocidib and 5-AZA effects in n=45 high-risk MDS patients. Our data revealed additive cytotoxic effects of the combination treatment. Mutational profiling of MDS samples identified ASXL1 mutations as predictors of response. Further, increased response rates were associated with higher gene-expression of the pro-apoptotic factor NOXA in ASXL1 mutated samples. The higher sensitivity of ASXL1 mutant cells to the combination treatment was confirmed in vivo in ASXL1Y588X transgenic mice. Overall, our study demonstrated augmented activity for the alvocidib + 5-AZA combination in higher-risk MDS and identified ASXL1 mutations as a biomarker of response for potential stratification studies.
RESUMEN
Lineage-defining transcription factors (TFs) are compelling targets for leukemia therapy, yet they are among the most challenging proteins to modulate directly with small molecules. We previously used CRISPR screening to identify a salt-inducible kinase 3 (SIK3) requirement for the growth of acute myeloid leukemia (AML) cell lines that overexpress the lineage TF myocyte enhancer factor (MEF2C). In this context, SIK3 maintains MEF2C function by directly phosphorylating histone deacetylase 4 (HDAC4), a repressive cofactor of MEF2C. In this study, we evaluated whether inhibition of SIK3 with the tool compound YKL-05-099 can suppress MEF2C function and attenuate disease progression in animal models of AML. Genetic targeting of SIK3 or MEF2C selectively suppressed the growth of transformed hematopoietic cells under in vitro and in vivo conditions. Similar phenotypes were obtained when cells were exposed to YKL-05-099, which caused cell-cycle arrest and apoptosis in MEF2C-expressing AML cell lines. An epigenomic analysis revealed that YKL-05-099 rapidly suppressed MEF2C function by altering the phosphorylation state and nuclear localization of HDAC4. Using a gatekeeper allele of SIK3, we found that the antiproliferative effects of YKL-05-099 occurred through on-target inhibition of SIK3 kinase activity. Based on these findings, we treated 2 different mouse models of MLL-AF9 AML with YKL-05-099, which attenuated disease progression in vivo and extended animal survival at well-tolerated doses. These findings validate SIK3 as a therapeutic target in MEF2C-addicted AML and provide a rationale for developing druglike inhibitors of SIK3 for definitive preclinical investigation and for studies in human patients.
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Compuestos de Anilina/farmacología , Leucemia Mieloide Aguda/prevención & control , Factores de Transcripción MEF2/metabolismo , Piperidinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/química , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Piridinas/farmacología , Pirimidinas/farmacología , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Apoptosis , Ciclo Celular , Proliferación Celular , Femenino , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Factores de Transcripción MEF2/genética , Ratones , Ratones Endogámicos C57BL , Tasa de Supervivencia , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Although great advances have been made in understanding the pathobiology of mixed lineage leukemia-rearranged (MLL-r) leukemias, therapies for this leukemia have remained limited, and clinical outcomes remain bleak. In order to identify novel targets for immunotherapy treatments, we compiled a lineage-independent MLL-r leukemia gene signature using publicly available data sets. Data from large leukemia repositories were filtered through the in silico human surfaceome, providing a list of highly predicted cell surface proteins overexpressed in MLL-r leukemias. LAMP5, a lysosomal associated membrane protein, is expressed highly and specifically in MLL-r leukemia. We found that LAMP5 is a direct target of the oncogenic MLL-fusion protein. LAMP5 depletion significantly inhibited leukemia cell growth in vitro and in vivo. Functional studies showed that LAMP-5 is a novel modulator of innate-immune pathways in MLL-r leukemias. Downregulation of LAMP5 led to inhibition of NF-kB signaling and increased activation of type-1 interferon signaling downstream of Toll-like receptor/interleukin 1 receptor activation. These effects were attributable to the critical role of LAMP-5 in transferring the signal flux from interferon signaling endosomes to pro-inflammatory signaling endosomes. Depletion of IRF7 was able to partially rescue the cell growth inhibition upon LAMP5 downregulation. Lastly, LAMP-5 was readily detected on the surface of MLL-r leukemia cells. Targeting surface LAMP-5 using an antibody-drug conjugate leads to significant cell viability decrease specifically in MLL-r leukemias. Overall, based on the limited expression throughout human tissues, we postulate that LAMP-5 could potentially serve as an immunotherapeutic target with a wide therapeutic window to treat MLL-r leukemias.
Asunto(s)
Leucemia Bifenotípica Aguda , Leucemia , N-Metiltransferasa de Histona-Lisina/genética , Humanos , Inmunoterapia , Leucemia/genética , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteína de la Leucemia Mieloide-Linfoide/metabolismoRESUMEN
BACKGROUND: Anaphylaxis is classically mediated by allergen cross-linking of IgE bound to the α chain of FcεRI, the mast cell/basophil high affinity IgE receptor. Allergen cross-linking of the IgE/FcεRI complex activates these cells, inducing release of disease-causing mediators, cytokines, and enzymes. We previously demonstrated that IgE-mediated anaphylaxis could be safely prevented in wild-type BALB/c mice by rapid desensitization with anti-mouse FcεRIα mAb. OBJECTIVE: This study sought to use humanized mice to extend these results to humans. METHODS: We actively immunized huFcεRIα/F709 mice, which express human (hu) instead of mouse FcεRIα and a mutant IL-4 receptor that lacks inhibitory function. We passively immunized huFcεRIα mice, as well as human cord blood-reconstituted reNSGS mice, which are immune-deficient, produce mast cell-stimulating human cytokines, and develop numerous human mast cells. For desensitization, we used anti-huFcεRIα mAbs that bind FcεRIα regardless of its association with IgE (noncompeting mAbs), and/or mAbs that compete with IgE for huFcεRIα binding (competing mAbs). Anaphylaxis was induced by intravenous injection of antigen or anti-huIgE mAb. RESULTS: Anti-huFcεRIα mAb rapid desensitization was safer and more effective than allergen rapid desensitization and suppressed anaphylaxis more rapidly than omalizumab or ligelizumab. Rapid desensitization of naïve, IgE-sensitized huFcεRIα mice and huFcεRIα/F709 mice that were egg-allergic with anti-FcεRIα mAbs safely removed >98% of IgE from peritoneal mast cells and completely suppressed IgE-mediated anaphylaxis. Rapid desensitization of reNSGS mice with anti-FcεRIα mAbs also safely removed â¼98% of mast cell IgE and prevented IgE-mediated anaphylaxis. CONCLUSIONS: Rapid desensitization with anti-FcεRIα mAbs may be a safe, effective, and practical way to prevent IgE-mediated anaphylaxis.
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Anafilaxia/inmunología , Anticuerpos Monoclonales/farmacología , Desensibilización Inmunológica/métodos , Receptores de IgE/antagonistas & inhibidores , Anafilaxia/prevención & control , Animales , Humanos , Ratones , Ratones Endogámicos BALB CRESUMEN
As a central regulator of cell polarity, the activity of CDC42 GTPase is tightly controlled in maintaining normal hematopoietic stem and progenitor cell (HSC/P) functions. We found that transformation of HSC/P to acute myeloid leukemia (AML) is associated with increased CDC42 expression and activity in leukemia cells. In a mouse model of AML, the loss of Cdc42 abrogates MLL-AF9-induced AML development. Furthermore, genetic ablation of CDC42 in both murine and human MLL-AF9 (MA9) cells decreased survival and induced differentiation of the clonogenic leukemia-initiating cells. We show that MLL-AF9 leukemia cells maintain cell polarity in the context of elevated Cdc42-guanosine triphosphate activity, similar to nonmalignant, young HSC/Ps. The loss of Cdc42 resulted in a shift to depolarized AML cells that is associated with a decrease in the frequency of symmetric and asymmetric cell divisions producing daughter cells capable of self-renewal. Importantly, we demonstrate that inducible CDC42 suppression in primary human AML cells blocks leukemia progression in a xenograft model. Thus, CDC42 loss suppresses AML cell polarity and division asymmetry, and CDC42 constitutes a useful target to alter leukemia-initiating cell fate for differentiation therapy.
Asunto(s)
Diferenciación Celular , División Celular , Polaridad Celular , Leucemia Mieloide Aguda/patología , Proteína de Unión al GTP cdc42/metabolismo , Animales , Carcinogénesis/genética , Carcinogénesis/patología , Diferenciación Celular/genética , División Celular/genética , Línea Celular Tumoral , Polaridad Celular/genética , Transformación Celular Neoplásica/patología , Células Clonales , Análisis Citogenético , Pruebas con Sangre Seca , GTP Fosfohidrolasas/metabolismo , Eliminación de Gen , Regulación Leucémica de la Expresión Génica , Células Madre Hematopoyéticas/metabolismo , Humanos , Leucemia Mieloide Aguda/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Células Madre Neoplásicas/patología , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Proteína de Unión al GTP cdc42/genéticaRESUMEN
Chromosomal translocation 8;21 is found in 40% of the FAB M2 subtype of acute myeloid leukemia (AML). The resultant in-frame fusion protein AML1-ETO (AE) acts as an initiating oncogene for leukemia development. AE immortalizes human CD34(+) cord blood cells in long-term culture. We assessed the transforming properties of the alternatively spliced AE isoform AE9a (or alternative splicing at exon 9), which is fully transforming in a murine retroviral model, in human cord blood cells. Full activity was realized only upon increased fusion protein expression. This effect was recapitulated in the AE9a murine AML model. Cotransduction of AE and AE9a resulted in a strong selective pressure for AE-expressing cells. In the context of AE, AE9a did not show selection for increased expression, affirming observations of human t(8;21) patient samples where full-length AE is the dominant protein detected. Mechanistically, AE9a showed defective transcriptional regulation of AE target genes that was partially corrected at high expression. Together, these results bring an additional perspective to our understanding of AE function and highlight the contribution of oncogene expression level in t(8;21) experimental models.
Asunto(s)
Transformación Celular Neoplásica , Subunidad alfa 2 del Factor de Unión al Sitio Principal/fisiología , Leucemia Mieloide Aguda/etiología , Proteínas de Fusión Oncogénica/fisiología , Proteína 1 Compañera de Translocación de RUNX1/fisiología , Animales , Células Cultivadas , Cromosomas Humanos Par 21 , Cromosomas Humanos Par 8 , Subunidad alfa 2 del Factor de Unión al Sitio Principal/análisis , Modelos Animales de Enfermedad , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Ratones , Proteínas de Fusión Oncogénica/análisis , Isoformas de Proteínas , Proteína 1 Compañera de Translocación de RUNX1/análisis , Translocación GenéticaRESUMEN
The CD44 gene encodes several protein isoforms due to alternative splicing and post translational modifications. Given that CD44 variant isoform 9 (CD44v9) is expressed within Spasmolytic Polypeptide/TFF2-Expressing Metaplasia (SPEM) glands during repair, CD44v9 may be play a funcitonal role during the process of regeneration of the gastric epithelium. Here we hypothesize that CD44v9 marks a regenerative cell lineage responsive to infiltrating macrophages during regeneration of the gastric epithelium. Ulcers were induced in CD44-deficient (CD44KO) and C57BL/6 (BL6) mice by a localized application of acetic acid to the serosal surface of the stomach. Gastric organoids expressing CD44v9 were derived from mouse stomachs and transplanted at the ulcer site of CD44KO mice. Ulcers, CD44v9 expression, proliferation and histology were measured 1, 3, 5 and 7-days post-injury. Human-derived gastric organoids were generated from stomach tissue collected from elderly (>55 years) or young (14-20 years) patients. Organoids were transplanted into the stomachs of NOD scid gamma (NSG) mice at the site of injury. Gastric injury was induced in NRG-SGM3 (NRGS) mice harboring human-derived immune cells (hnNRGS) and the immune profile anlayzed by CyTOF. CD44v9 expression emerged within regenerating glands the ulcer margin in response to injury. While ulcers in BL6 mice healed within 7-days post-injury, CD44KO mice exhibited loss of repair and epithelial regeneration. Ulcer healing was promoted in CD44KO mice by transplanted CD55v9-expressing gastric organoids. NSG mice exhibited loss of CD44v9 expression and gastric repair. Transplantation of human-derived gastric organoids from young, but not aged stomachs promoted repair in NSG mouse stomachs in response to injury. Finally, compared to NRGS mice, huNRGS animals exhibited reduced ulcer sizes, an infiltration of human CD162+ macrophages and an emergence of CD44v9 expression in SPEM. Thus, during repair of the gastic epithelium CD44v9 emerges within a regenerative cell lineage that coincides with macrophage inflitration within the injured mucosa. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Asunto(s)
Mucosa Gástrica/fisiología , Receptores de Hialuranos/genética , Regeneración/fisiología , Úlcera Gástrica/metabolismo , Adolescente , Factores de Edad , Anciano , Animales , Células Cultivadas , Mucosa Gástrica/patología , Variación Genética/fisiología , Humanos , Receptores de Hialuranos/metabolismo , Receptores de Hialuranos/fisiología , Macrófagos/fisiología , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Noqueados , Persona de Mediana Edad , Organoides/citología , Organoides/trasplante , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/fisiología , Regeneración/genética , Úlcera Gástrica/genética , Úlcera Gástrica/patología , Cicatrización de Heridas/fisiología , Adulto JovenRESUMEN
Acute myeloid leukemia (AML) is associated with a poor survival rate, and there is an urgent need for novel and more efficient therapies, ideally targeting AML stem cells that are essential for maintaining the disease. The interleukin 1 receptor accessory protein (IL1RAP; IL1R3) is expressed on candidate leukemic stem cells in the majority of AML patients, but not on normal hematopoietic stem cells. We show here that monoclonal antibodies targeting IL1RAP have strong antileukemic effects in xenograft models of human AML. We demonstrate that effector-cell-mediated killing is essential for the observed therapeutic effects and that natural killer cells constitute a critical human effector cell type. Because IL-1 signaling is important for the growth of AML cells, we generated an IL1RAP-targeting antibody capable of blocking IL-1 signaling and show that this antibody suppresses the proliferation of primary human AML cells. Hence, IL1RAP can be efficiently targeted with an anti-IL1RAP antibody capable of both achieving antibody-dependent cellular cytotoxicity and blocking of IL-1 signaling as modes of action. Collectively, these results provide important evidence in support of IL1RAP as a target for antibody-based treatment of AML.
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Anticuerpos Monoclonales/uso terapéutico , Proteína Accesoria del Receptor de Interleucina-1/antagonistas & inhibidores , Leucemia Mieloide Aguda/tratamiento farmacológico , Proteínas de Neoplasias/antagonistas & inhibidores , Animales , Citotoxicidad Celular Dependiente de Anticuerpos , División Celular , Línea Celular Tumoral , Citotoxicidad Inmunológica , Humanos , Interleucina-1/antagonistas & inhibidores , Proteína Accesoria del Receptor de Interleucina-1/inmunología , Células Asesinas Naturales/inmunología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Proteínas de Neoplasias/inmunología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The model systems available for studying human hematopoiesis, malignant hematopoiesis, and hematopoietic stem cell (HSC) function in vivo have improved dramatically over the last decade, primarily due to improvements in xenograft mouse strains. Several recent reviews have focused on the historic development of immunodeficient mice over the last 2 decades, as well as their use in understanding human HSC and leukemia stem cell (LSC) biology and function in the context of a humanized mouse. However, in the intervening time since these reviews, a number of new mouse models, technical approaches, and scientific advances have been made. In this review, we update the reader on the newest and best models and approaches available for studying human malignant and normal HSCs in immunodeficient mice, including newly developed mice for use in chemotherapy testing and improved techniques for humanizing mice without laborious purification of HSC. We also review some relevant scientific findings from xenograft studies and highlight the continued limitations that confront researchers working with human HSC and LSC in vivo.
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Trasplante de Células Madre Hematopoyéticas/métodos , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/patología , Células Madre Neoplásicas/citología , Células Madre Neoplásicas/patología , Animales , Modelos Animales de Enfermedad , Hematopoyesis , Células Madre Hematopoyéticas/metabolismo , Humanos , Leucemia/metabolismo , Leucemia/patología , Ratones , Trasplante de Neoplasias/métodos , Células Madre Neoplásicas/metabolismo , Trasplante Heterólogo/métodosRESUMEN
Leukemias with MLL translocations are often found in infants and are associated with poor outcomes. The pathogenesis of MLL-fusion leukemias has been linked to upregulation of HOX/MEIS1 genes. The functions of the Hox/Meis1 complex in leukemia, however, remain elusive. Here, we used inducible Meis1-knockout mice coupled with MLL-AF9 knockin mice to decipher the mechanistic role of Meis1 in established MLL leukemia. We demonstrate that Meis1 is essential for maintenance of established leukemia. In addition, in both the murine model and human leukemia cells, we found that Meis1 loss led to increased oxidative stress, oxygen flux, and apoptosis. Gene expression and chromatin immunoprecipitation studies revealed hepatic leukemia factor (HLF) as a target gene of Meis1. Hypoxia or HLF expression reversed the oxidative stress, rescuing leukemia development in Meis1-deficient cells. Thus, the leukemia-promoting properties of Meis1 are at least partly mediated by a low-oxidative state, aided by HLF. These results suggest that stimulants of oxidative metabolism could have therapeutic potential in leukemia treatment.
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Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Proteínas de Homeodominio/metabolismo , Leucemia/metabolismo , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Estrés Oxidativo , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Western Blotting , Hipoxia de la Célula , Línea Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Ácido Dicloroacético/farmacología , Regulación Leucémica de la Expresión Génica , Células HEK293 , Proteínas de Homeodominio/genética , Humanos , Leucemia/genética , Leucemia/patología , Ratones Noqueados , Ratones Transgénicos , Proteína 1 del Sitio de Integración Viral Ecotrópica Mieloide , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteínas de Neoplasias/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas de Fusión Oncogénica/genética , Fosforilación Oxidativa/efectos de los fármacos , Interferencia de ARN , Especies Reactivas de Oxígeno/metabolismo , TranscriptomaRESUMEN
Epigenetic pathways can regulate gene expression by controlling and interpreting chromatin modifications. Cancer cells are characterized by altered epigenetic landscapes, and commonly exploit the chromatin regulatory machinery to enforce oncogenic gene expression programs. Although chromatin alterations are, in principle, reversible and often amenable to drug intervention, the promise of targeting such pathways therapeutically has been limited by an incomplete understanding of cancer-specific dependencies on epigenetic regulators. Here we describe a non-biased approach to probe epigenetic vulnerabilities in acute myeloid leukaemia (AML), an aggressive haematopoietic malignancy that is often associated with aberrant chromatin states. By screening a custom library of small hairpin RNAs (shRNAs) targeting known chromatin regulators in a genetically defined AML mouse model, we identify the protein bromodomain-containing 4 (Brd4) as being critically required for disease maintenance. Suppression of Brd4 using shRNAs or the small-molecule inhibitor JQ1 led to robust antileukaemic effects in vitro and in vivo, accompanied by terminal myeloid differentiation and elimination of leukaemia stem cells. Similar sensitivities were observed in a variety of human AML cell lines and primary patient samples, revealing that JQ1 has broad activity in diverse AML subtypes. The effects of Brd4 suppression are, at least in part, due to its role in sustaining Myc expression to promote aberrant self-renewal, which implicates JQ1 as a pharmacological means to suppress MYC in cancer. Our results establish small-molecule inhibition of Brd4 as a promising therapeutic strategy in AML and, potentially, other cancers, and highlight the utility of RNA interference (RNAi) screening for revealing epigenetic vulnerabilities that can be exploited for direct pharmacological intervention.
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Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Proteínas Nucleares/metabolismo , Interferencia de ARN , Factores de Transcripción/metabolismo , Acetilación , Animales , Azepinas/farmacología , Diferenciación Celular , Línea Celular Tumoral , Proliferación Celular , Cromatina/metabolismo , Progresión de la Enfermedad , Epigénesis Genética/genética , Regulación Neoplásica de la Expresión Génica , Genes myc/genética , Histonas/metabolismo , Humanos , Leucemia Mieloide Aguda/patología , Ratones , Trasplante de Neoplasias , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/patología , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/genética , ARN Interferente Pequeño/genética , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética , Triazoles/farmacologíaRESUMEN
Immunodeficient mice are now readily engrafted with human hematopoietic cells. However, these mice are susceptible to graft-versus-host disease (GVHD) induced by the engraftment and rapid expansion of coinjected human T cells. Therefore, highly purified sample populations must be used, adding significant time, expense, and effort. Here, we have explored in vivo and in vitro methods utilizing anti-T-cell antibodies to circumvent this problem. Intraperitoneal injection of the antibody within 48 hours prevented GVHD. Alternatively, short-term in vitro incubation of cells with antibody immediately before transplant was equally effective. Although in vitro antithymocyte globulin treatment resulted in a dramatic loss of SCID-repopulating cells (SRCs), treatment with OKT3 or UCHT1 abrogated GVHD risk and preserved engraftment potential. Leukemia samples that presented with substantial human T-cell contamination were effectively rescued from GVHD. In addition, OKT3 treatment of unfractionated cord blood resulted in robust engraftment of primary and secondary mice that was indistinguishable from grafts obtained using purified CD34(+) cells. Limiting dilution analysis of unfractionated blood demonstrated a SRC frequency of 1 in 300 to 500 CD34(+) cells, similar to that of purified hematopoietic stem and progenitor cells. This protocol streamlines xenograft studies while significantly reducing the cost and time of the procedure.
Asunto(s)
Enfermedad Injerto contra Huésped/prevención & control , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Inmunosupresores/uso terapéutico , Muromonab-CD3/uso terapéutico , Adulto , Animales , Eliminación de Componentes Sanguíneos , Células Cultivadas , Evaluación Preclínica de Medicamentos , Femenino , Enfermedad Injerto contra Huésped/etiología , Xenoinjertos , Humanos , Masculino , Ratones , Ratones SCID , Ratones Transgénicos , Linfocitos T/efectos de los fármacos , Linfocitos T/fisiología , Acondicionamiento Pretrasplante/métodosRESUMEN
MicroRNAs (miRNAs), small noncoding RNAs that regulate target gene mRNAs, are known to contribute to pathogenesis of cancers. Acute myeloid leukemia (AML) is a group of heterogeneous hematopoietic malignancies with various chromosomal and/or molecular abnormalities. AML with chromosomal translocations involving the mixed lineage leukemia (MLL) gene are usually associated with poor survival. In the present study, through a large-scale, genomewide miRNA expression assay, we show that microRNA-9 (miR-9) is the most specifically up-regulated miRNA in MLL-rearranged AML compared with both normal control and non-MLL-rearranged AML. We demonstrate that miR-9 is a direct target of MLL fusion proteins and can be significantly up-regulated in expression by the latter in human and mouse hematopoietic stem/progenitor cells. Depletion of endogenous miR-9 expression by an appropriate antagomiR can significantly inhibit cell growth/viability and promote apoptosis in human MLL-rearranged AML cells, and the opposite is true when expression of miR-9 is forced. Blocking endogenous miR-9 function by anti-miRNA sponge can significantly inhibit, whereas forced expression of miR-9 can significantly promote, MLL fusion-induced immortalization/transformation of normal mouse bone marrow progenitor cells in vitro. Furthermore, forced expression of miR-9 can significantly promote MLL fusion-mediated leukemogenesis in vivo. In addition, a group of putative target genes of miR-9 exhibited a significant inverse correlation of expression with miR-9 in a series of leukemia sample sets, suggesting that they are potential targets of miR-9 in MLL-rearranged AML. Collectively, our data demonstrate that miR-9 is a critical oncomiR in MLL-rearranged AML and can serve as a potential therapeutic target to treat this dismal disease.
Asunto(s)
Leucemia Mieloide Aguda/genética , MicroARNs/fisiología , Proteína de la Leucemia Mieloide-Linfoide/genética , Apoptosis/genética , Supervivencia Celular/genética , Proteínas de Unión al ADN/fisiología , Humanos , Leucemia Mieloide Aguda/patología , Proteína del Locus del Complejo MDS1 y EV11 , MicroARNs/genética , Proto-Oncogenes/fisiología , Factores de Transcripción/fisiologíaRESUMEN
The ten-eleven translocation 1 (TET1) gene is the founding member of the TET family of enzymes (TET1/2/3) that convert 5-methylcytosine to 5-hydroxymethylcytosine. Although TET1 was first identified as a fusion partner of the mixed lineage leukemia (MLL) gene in acute myeloid leukemia carrying t(10,11), its definitive role in leukemia is unclear. In contrast to the frequent down-regulation (or loss-of-function mutations) and critical tumor-suppressor roles of the three TET genes observed in various types of cancers, here we show that TET1 is a direct target of MLL-fusion proteins and is significantly up-regulated in MLL-rearranged leukemia, leading to a global increase of 5-hydroxymethylcytosine level. Furthermore, our both in vitro and in vivo functional studies demonstrate that Tet1 plays an indispensable oncogenic role in the development of MLL-rearranged leukemia, through coordination with MLL-fusion proteins in regulating their critical cotargets, including homeobox A9 (Hoxa9)/myeloid ecotropic viral integration 1 (Meis1)/pre-B-cell leukemia homeobox 3 (Pbx3) genes. Collectively, our data delineate an MLL-fusion/Tet1/Hoxa9/Meis1/Pbx3 signaling axis in MLL-rearranged leukemia and highlight TET1 as a potential therapeutic target in treating this presently therapy-resistant disease.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Regulación Neoplásica de la Expresión Génica/fisiología , Leucemia Mieloide Aguda/metabolismo , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Transducción de Señal/fisiología , 5-Metilcitosina/análogos & derivados , Inmunoprecipitación de Cromatina , Cromatografía Liquida , Citosina/análogos & derivados , Citosina/metabolismo , Perfilación de la Expresión Génica , N-Metiltransferasa de Histona-Lisina , Proteínas de Homeodominio/metabolismo , Humanos , Immunoblotting , Análisis por Micromatrices , Oxigenasas de Función Mixta , Proteína 1 del Sitio de Integración Viral Ecotrópica Mieloide , Proteínas de Neoplasias/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Transducción de Señal/genética , Espectrometría de Masas en TándemRESUMEN
We designed ROS-activated cytotoxic agents (RACs) that are active against AML cancer cells. In this study, the mechanism of action and synergistic effects against cells coexpressing the AML oncogenes MLL-AF9 fusion and FLT3-ITD were investigated. One RAC (RAC1) had an IC50 value of 1.8±0.3â µm, with ninefold greater selectivity for transformed cells compared to untransformed cells. Treatment induced DNA strand breaks, apoptosis, and cell cycle arrest. Proteomics and transcriptomics revealed enhanced expression of the pentose phosphate pathway, DNA repair, and pathways common to cell stress. Western blotting confirmed repair by homologous recombination. Importantly, RAC1 treatment was synergistic in combination with multiple pathway-targeting therapies in AML cells but less so in untransformed cells. Together, these results demonstrate that RAC1 can selectively target poor prognosis AML and that it does so by creating DNA double-strand breaks that require homologous recombination.
Asunto(s)
Compuestos de Anilina/química , Antineoplásicos/farmacología , ADN/metabolismo , Fenol/química , Reparación del ADN por Recombinación/efectos de los fármacos , Compuestos de Anilina/toxicidad , Antineoplásicos/química , Apoptosis/efectos de los fármacos , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Cromatografía Líquida de Alta Presión , ADN/química , Roturas del ADN de Doble Cadena/efectos de los fármacos , Humanos , Proteínas Nucleares/metabolismo , Fenol/toxicidad , Proteómica , Especies Reactivas de Oxígeno/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
As acute myeloid leukemia (AML) xenograft models improve, the potential for using them to evaluate novel therapeutic strategies becomes more appealing. Currently, there is little information on using standard chemotherapy regimens in AML xenografts. Here we have characterized the immunodeficient mouse response to combined Ara-C (cytarabine) and doxorubicin treatment. We observed significant toxicity associated with doxorubicin that required optimization of the route of injection as well as the maximum-tolerated dose for immunodeficient strains. Mice treated with an optimized 5-day induction protocol showed transient weight loss, short-term reduction of peripheral blood cell and platelet counts, and slight anemia. Considerable cytotoxicity was observed in the bone marrow (BM), with primitive LSK cells having a significant survival advantage relative to more mature cells, consistent with the idea of chemotherapy targeting actively growing cells. Treated leukemic mice demonstrated reduced disease burden and increased survival, demonstrating efficacy. AML cells showed significantly increased sensitivity to doxorubicin-containing therapy compared with murine BM cells. Although early treatment could result in some cures, mice with significant leukemia grafts were not cured by using induction therapy alone. Overall, the data show that this model system is useful for the evaluation of novel chemotherapies in combination with standard induction therapy.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Resistencia a Antineoplásicos , Leucemia Mieloide Aguda/tratamiento farmacológico , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Línea Celular Tumoral , Citarabina/administración & dosificación , Doxorrubicina/administración & dosificación , Vías de Administración de Medicamentos , Resistencia a Antineoplásicos/fisiología , Hematopoyesis/efectos de los fármacos , Humanos , Leucemia Mieloide Aguda/sangre , Leucemia Mieloide Aguda/patología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones SCID , Ratones Transgénicos , Resultado del TratamientoRESUMEN
Acute myeloid leukemia (AML) is a heterogeneous group of hematopoietic malignancies with variable response to treatment. AMLs bearing MLL (mixed lineage leukemia) rearrangements are associated with intermediate or poor survival. MicroRNAs (miRNAs), a class of small noncoding RNAs, have been postulated to be important gene expression regulators virtually in all biological processes, including leukemogenesis. Through a large-scale, genome-wide miRNA expression profiling assay of 85 human AML and 15 normal control samples, we show that among 48 miRNAs that are significantly differentially expressed between MLL- and non-MLL-rearranged AML samples, only one (miR-495) is expressed at a lower level in MLL-rearranged AML than in non-MLL-rearranged AML; meanwhile, miR-495 is also significantly down-regulated in MLL-rearranged AML samples compared with normal control samples. Through in vitro colony-forming/replating assays and in vivo bone marrow transplantation studies, we show that forced expression of miR-495 significantly inhibits MLL-fusion-mediated cell transformation in vitro and leukemogenesis in vivo. In human leukemic cells carrying MLL rearrangements, ectopic expression of miR-495 greatly inhibits cell viability and increases cell apoptosis. Furthermore, our studies demonstrate that PBX3 and MEIS1 are two direct target genes of miR-495, and forced expression of either of them can reverse the effects of miR-495 overexpression on inhibiting cell viability and promoting apoptosis of human MLL-rearranged leukemic cells. Thus, our data indicate that miR-495 likely functions as a tumor suppressor in AML with MLL rearrangements by targeting essential leukemia-related genes.