RESUMEN
Bortezomib (BTZ) was recently evaluated in a randomized phase 3 clinical trial by the Children's Oncology Group (COG) that compared standard chemotherapy (cytarabine, daunorubicin, and etoposide [ADE]) vs standard therapy with BTZ (ADEB) for de novo pediatric acute myeloid leukemia (AML). Although the study concluded that BTZ did not improve outcome overall, we examined patient subgroups benefiting from BTZ-containing chemotherapy using proteomic analyses. The proteasome inhibitor BTZ disrupts protein homeostasis and activates cytoprotective heat shock responses. Total heat shock factor 1 (HSF1) and phosphorylated HSF1 (HSF1-pSer326) were measured in leukemic cells from 483 pediatric patients using reverse phase protein arrays. HSF1-pSer326 phosphorylation was significantly lower in pediatric AML compared with CD34+ nonmalignant cells. We identified a strong correlation between HSF1-pSer326 expression and BTZ sensitivity. BTZ significantly improved outcome of patients with low-HSF1-pSer326 with a 5-year event-free survival of 44% (ADE) vs 67% for low-HSF1-pSer326 treated with ADEB (P = .019). To determine the effect of HSF1 expression on BTZ potency in vitro, cell viability with HSF1 gene variants that mimicked phosphorylated (S326A) and nonphosphorylated (S326E) HSF1-pSer326 were examined. Those with increased HSF1 phosphorylation showed clear resistance to BTZ vs those with wild-type or reduced HSF1-phosphorylation. We hypothesize that HSF1-pSer326 expression could identify patients who benefit from BTZ-containing chemotherapy.
Asunto(s)
Antineoplásicos/uso terapéutico , Bortezomib/uso terapéutico , Factores de Transcripción del Choque Térmico/genética , Leucemia Mieloide Aguda/tratamiento farmacológico , Niño , Preescolar , Resistencia a Antineoplásicos , Femenino , Humanos , Lactante , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/genética , Masculino , Mutación Puntual , Pronóstico , TranscriptomaRESUMEN
FMS-like Tyrosine Kinase 3 (FLT3) mutation is associated with poor survival in acute myeloid leukemia (AML). The specific Anexelekto/MER Tyrosine Kinase (AXL) inhibitor, ONO-7475, kills FLT3-mutant AML cells with targets including Extracellular- signal Regulated Kinase (ERK) and Myeloid Cell Leukemia 1 (MCL1). ERK and MCL1 are known resistance factors for Venetoclax (ABT-199), a popular drug for AML therapy, prompting the investigation of the efficacy of ONO-7475 in combination with ABT-199 in vitro and in vivo. ONO-7475 synergizes with ABT-199 to potently kill FLT3-mutant acute myeloid leukemia cell lines and primary cells. ONO-7475 is effective against ABT-199-resistant cells including cells that overexpress MCL1. Proteomic analyses revealed that ABT-199-resistant cells expressed elevated levels of pro-growth and anti-apoptotic proteins compared to parental cells, and that ONO-7475 reduced the expression of these proteins in both the parental and ABT-199-resistant cells. ONO-7475 treatment significantly extended survival as a single in vivo agent using acute myeloid leukemia cell lines and PDX models. Compared to ONO-7474 monotherapy, the combination of ONO-7475/ABT-199 was even more potent in reducing leukemic burden and prolonging the survival of mice in both model systems. These results suggest that the ONO-7475/ABT-199 combination may be effective for AML therapy.
Asunto(s)
Resistencia a Antineoplásicos , Leucemia Mieloide Aguda , Inhibidores de Proteínas Quinasas , Tirosina Quinasa c-Mer , Animales , Apoptosis , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Línea Celular Tumoral , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Ratones , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteómica , Sulfonamidas/farmacología , Tirosina Quinasa c-Mer/antagonistas & inhibidores , Tirosina Quinasa 3 Similar a fms/genéticaRESUMEN
In acute myeloid leukemia (AML), high Galectin 3 (LGALS3) expression is associated with poor prognosis. The role of LGALS3 derived from mesenchymal stromal cells (MSC) in the AML microenvironment is unclear; however, we have recently found high LGALS3 expression in MSC derived from AML patients is associated with relapse. In this study, we used reverse phase protein analysis (RPPA) to correlate LGALS3 expression in AML MSC with 119 other proteins including variants of these proteins such as phosphorylated forms or cleaved forms to identify biologically relevant pathways. RPPA revealed that LGALS3 protein was positively correlated with expression of thirteen proteins including MYC, phosphorylated beta-Catenin (p-CTNNB1), and AKT2 and negatively correlated with expression of six proteins including integrin beta 3 (ITGB3). String analysis revealed that proteins positively correlated with LGALS3 showed strong interconnectivity. Consistent with the RPPA results, LGALS3 suppression by shRNA in MSC resulted in decreased MYC and AKT expression while ITGB3 was induced. In co-culture, the ability of AML cell to adhere to MSC LGALS3 shRNA transductants was reduced compared to AML cell adhesion to MSC control shRNA transductants. Finally, use of novel specific LGALS3 inhibitor CBP.001 in co-culture of AML cells with MSC reduced viable leukemia cell populations with induced apoptosis and augmented the chemotherapeutic effect of AraC. In summary, the current study demonstrates that MSC-derived LGALS3 may be critical for important biological pathways for MSC homeostasis and for regulating AML cell localization and survival in the leukemia microenvironmental niche.
Asunto(s)
Galectina 3/metabolismo , Leucemia Mieloide Aguda/metabolismo , Células Madre Mesenquimatosas/metabolismo , Regulación hacia Arriba , Proteínas Sanguíneas , Técnicas de Cocultivo , Galectinas , Regulación Neoplásica de la Expresión Génica , Humanos , Células Madre Mesenquimatosas/citología , Fosforilación , Mapas de Interacción de Proteínas , Proteómica , Células THP-1 , Células Tumorales Cultivadas , Microambiente TumoralRESUMEN
Autophagy is a cellular adaptive mechanism to stress, including that induced by chemotherapeutic agents. Reverse phase protein array suggested that high expression of the essential autophagy-related protein, Atg7, was associated with shorter remission in newly diagnosed acute myeloid leukemia (AML) patient samples, indicating a role in chemoresistance. Knockdown of Atg7 in AML cells using short hairpin RNA markedly increased apoptosis and DNA damage following treatment with cytarabine and idarubicin. Interestingly, coculture of AML cells with stromal cells increased autophagy and chemoresistance in the AML cells exposed to chemotherapeutic agents, and this was reversed following Atg7 knockdown. This effect was further enhanced by concomitant knockdown of Atg7 in both AML and stromal cells. These findings strongly suggest that Atg7, and likely microenvironment autophagy in general, plays an important role in AML chemoresistance. Mechanistic studies revealed that Atg7 knockdown induced a proapoptotic phenotype in AML cells, which was manifested by an increased NOXA expression at the transcriptional level. Finally, in a mouse model of human leukemia, Atg7 knockdown extended overall survival after chemotherapy. Thus, the inhibition of Atg7 appears to be a valid strategy to enhance chemosensitivity, and it could indeed improve outcomes in AML therapy.
Asunto(s)
Proteína 7 Relacionada con la Autofagia , Autofagia , Resistencia a Antineoplásicos , Leucemia Mieloide Aguda , Microambiente Tumoral , Animales , Autofagia/efectos de los fármacos , Autofagia/genética , Proteína 7 Relacionada con la Autofagia/genética , Proteína 7 Relacionada con la Autofagia/metabolismo , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Células HL-60 , Humanos , Células K562 , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Proteínas Proto-Oncogénicas c-bcl-2/genética , Células del Estroma/metabolismo , Células del Estroma/patología , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Mesenchymal stromal cells (MSC) support acute myeloid leukemia (AML) cell survival in the bone marrow (BM) microenvironment. Protein expression profiles of AML-derived MSC are unknown. Reverse phase protein array analysis was performed to compare expression of 151 proteins from AML-MSC (n=106) with MSC from healthy donors (n=71). Protein expression differed significantly between the two groups with 19 proteins over-expressed in leukemia stromal cells and 9 over-expressed in normal stromal cells. Unbiased hierarchical clustering analysis of the samples using these 28 proteins revealed three protein constellations whose variation in expression defined four MSC protein expression signatures: Class 1, Class 2, Class 3, and Class 4. These cell populations appear to have clinical relevance. Specifically, patients with Class 3 cells have longer survival and remission duration compared to other groups. Comparison of leukemia MSC at first diagnosis with those obtained at salvage (i.e. relapse/refractory) showed differential expression of 9 proteins reflecting a shift toward osteogenic differentiation. Leukemia MSC are more senescent compared to their normal counterparts, possibly due to the overexpressed p53/p21 axis as confirmed by high ß-galactosidase staining. In addition, overexpression of BCL-XL in leukemia MSC might give survival advantage under conditions of senescence or stress and overexpressed galectin-3 exerts profound immunosuppression. Together, our findings suggest that the identification of specific populations of MSC in AML patients may be an important determinant of therapeutic response.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Leucemia Mieloide Aguda/mortalidad , Células Madre Mesenquimatosas/metabolismo , Recurrencia Local de Neoplasia/mortalidad , Análisis por Matrices de Proteínas , Adulto , Estudios de Casos y Controles , Diferenciación Celular , Proliferación Celular , Femenino , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Masculino , Células Madre Mesenquimatosas/patología , Persona de Mediana Edad , Recurrencia Local de Neoplasia/metabolismo , Recurrencia Local de Neoplasia/patología , Pronóstico , Tasa de Supervivencia , Células Tumorales CultivadasRESUMEN
Galectin 3 is a member of a family of ß-galactoside binding proteins and has emerged as an important regulator of diverse functions critical in cancer biology including apoptosis, metastasis, immune surveillance, molecular trafficking, mRNA splicing, gene expression, and inflammation. Galectin 3's ability to support cancer cell survival by intra-cellular and extra-cellular mechanisms suggests this molecule is an important component of the tumor microenvironment that potentially could be targeted for therapy. Data is emerging that Galectin 3 is elevated in many cancers including solid tumors and the cancers of the blood. Galectin 3 also appears to be a key molecule produced by tumor microenvironment support cells including mesenchymal stromal cells (MSC) to suppress immune surveillance by killing T cells and interfering with NK cell function and by supporting metastasis. Levels of Galectin 3 increase in the MSC of aging mice and perhaps this contributes to the development of cancer in the elderly. Galectin 3 modulates surface protein expression of a diverse set of glycoproteins including CD44 by regulating endocytosis of these proteins. In addition, Galectin 3 binding to receptor kinases such as CD45 and the T cell receptor is critical in the regulation of their function. In this review I will examine the various mechanisms how Galectin 3 supports chemoresistance and metastasis in solid tumors and in leukemia and lymphoma. I will also discuss possible therapeutic strategies to target this Galectin for cancer therapy. This article is part of a Special Issue entitled: Tumor Microenvironment Regulation of Cancer Cell Survival, Metastasis, Inflammation, and Immune Surveillance edited by Peter Ruvolo and Gregg L. Semenza.
Asunto(s)
Apoptosis/inmunología , Galectina 3/inmunología , Neoplasias/inmunología , Microambiente Tumoral/inmunología , Antineoplásicos/uso terapéutico , Adhesión Celular/inmunología , Galectina 3/antagonistas & inhibidores , Galectina 3/metabolismo , Humanos , Vigilancia Inmunológica/inmunología , Modelos Inmunológicos , Terapia Molecular Dirigida , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Microambiente Tumoral/efectos de los fármacosRESUMEN
Galectin 3 (LGALS3) expression is prognostic for poor survival in acute myeloid leukemia (AML) patients. GCS-100 is a novel galectin inhibitor that may prove useful for AML therapy. In this study, we found that GCS-100 induced apoptosis in AML cells. The agent reduced MCL-1 expression suggesting that GCS-100 could be more effective when combined with a BH3 mimetic. Indeed, potent synergistic cytotoxicity was achieved when GCS-100 was combined with ABT-737 or ABT-199. Furthermore, the GCS-100/ABT-199 combination was effective against primary AML blast cells from patients with FLT3 ITD mutations, which is another prognostic factor for poor outcome in AML. This activity may involve wild-type p53 as shRNA knockdown of LGALS3 or galectin 1 (LGALS1) sensitized wild-type p53 OCI-AML3 cells to GCS-100/ABT-737-induced apoptosis to a much greater extent than p53 null THP-1 cells. Suppression of LGALS3 by shRNA inhibited MCL-1 expression in OCI-AML3 cells, but not THP-1 cells, suggesting the induced sensitivity to ABT-737 may involve a MCL-1 mediated mechanism. OCI-AML3 cells with LGALS1 shRNA were also sensitized to ABT-737. However, these cells exhibited increased MCL-1 expression, so MCL-1 reduction is apparently not required in this process. A role for p53 appears important as GCS-100 induces p53 expression and shRNA knockdown of p53 protected OCI-AML3 cells from the cytotoxic effects of the GCS-100/ABT-737 treatment combination. Our results suggest that galectins regulate a survival axis in AML cells, which may be targeted via combined inhibition with drugs such as GCS-100 and ABT-199.
Asunto(s)
Compuestos de Bifenilo/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Leucemia Mieloide Aguda/patología , Nitrofenoles/farmacología , Polisacáridos/farmacología , Sulfonamidas/farmacología , Proteína p53 Supresora de Tumor/genética , Anciano , Anciano de 80 o más Años , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Compuestos de Bifenilo/administración & dosificación , Compuestos Bicíclicos Heterocíclicos con Puentes/administración & dosificación , Línea Celular Tumoral , Sinergismo Farmacológico , Femenino , Galectinas/antagonistas & inhibidores , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Masculino , Persona de Mediana Edad , Nitrofenoles/administración & dosificación , Fragmentos de Péptidos/química , Piperazinas/administración & dosificación , Piperazinas/farmacología , Polisacáridos/administración & dosificación , Proteínas Proto-Oncogénicas/química , Sulfonamidas/administración & dosificaciónRESUMEN
Overexpression of antiapoptotic Bcl-2 proteins such as Bcl-2, Bcl-xL, and Mcl-1 is widely associated with tumor initiation, progression, and chemoresistance. Furthermore, it has been demonstrated that Mcl-1 upregulation renders several types of cancers resistant to the Bcl-2/Bcl-xL inhibitors ABT-737 and ABT-263. The emerging importance of Mcl-1 in pathogenesis and drug resistance makes it a high-priority therapeutic target. In this study, we showed that inhibition of Mcl-1 with a novel pan-Bcl-2 inhibitor (-)BI97D6 potently induced apoptosis in acute myeloid leukemia (AML) cells. (-)BI97D6 induced hallmarks of mitochondrial apoptosis, disrupted Mcl-1/Bim and Bcl-2/Bax interactions, and stimulated cell death via the Bak/Bax-dependent mitochondrial apoptosis pathway, suggesting on-target mechanisms. As a single agent, this pan-Bcl-2 inhibitor effectively overcame AML cell apoptosis resistance mediated by Mcl-1 or by interactions with bone marrow mesenchymal stromal cells. (-)BI97D6 was also potent in killing refractory primary AML cells. Importantly, (-)BI97D6 killed AML leukemia stem/progenitor cells while largely sparing normal hematopoietic stem/progenitor cells. These findings demonstrate that pan-Bcl-2 inhibition by an Mcl-1-targeting inhibitor not only overcomes intrinsic drug resistance ensuing from functional redundancy of Bcl-2 proteins, but also abrogates extrinsic resistance caused by the protective tumor microenvironment.
Asunto(s)
Apoptosis/efectos de los fármacos , Compuestos de Bifenilo/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Gosipol/análogos & derivados , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/patología , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/antagonistas & inhibidores , Naftoquinonas/farmacología , Nitrofenoles/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Sulfonamidas/farmacología , Animales , Western Blotting , Proliferación Celular/efectos de los fármacos , Femenino , Citometría de Flujo , Gosipol/farmacología , Humanos , Técnicas para Inmunoenzimas , Inmunoprecipitación , Leucemia Mieloide Aguda/metabolismo , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones SCID , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Piperazinas/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Células Madre/citología , Células Madre/efectos de los fármacos , Células Madre/metabolismo , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Nearly one-third of patients with acute myeloid leukemia have FMS-like tyrosine kinase 3 mutations and thus have poor survival prospects. Receptor tyrosine kinase anexelekto is critical for FMS-like tyrosine kinase 3 signaling and participates in FMS-like tyrosine kinase 3 inhibitor resistance mechanisms. Thus, strategies targeting anexelekto could prove useful for acute myeloid leukemia therapy. ONO-7475 is an inhibitor with high specificity for anexelekto and MER tyrosine kinase. Herein, we report that ONO-7475 potently arrested growth and induced apoptosis in acute myeloid leukemia with internal tandem duplication mutation of FMS-like tyrosine kinase 3. MER tyrosine kinase-lacking MOLM13 cells were sensitive to ONO-7475, while MER tyrosine kinase expressing OCI-AML3 cells were resistant, suggesting that the drug acts via anexelekto in acute myeloid leukemia cells. Reverse phase protein analysis of ONO-7475 treated cells revealed that cell cycle regulators like cyclin dependent kinase 1, cyclin B1, polo-like kinase 1, and retinoblastoma were suppressed. ONO-7475 suppressed cyclin dependent kinase 1, cyclin B1, polo-like kinase 1 gene expression suggesting that anexelekto may regulate the cell cycle, at least in part, via transcriptional mechanisms. Importantly, ONO-7475 was effective in a human FMS-like tyrosine kinase 3 with internal tandem duplication mutant murine xenograft model. Mice fed a diet containing ONO-7475 exhibited significantly longer survival and, interestingly, blocked leukemia cell infiltration in the liver. In summary, ONO-7475 effectively kills acute myeloid leukemia cells in vitro and in vivo by mechanisms that involve disruption of diverse survival and proliferation pathways.
Asunto(s)
Leucemia Mieloide Aguda/patología , Inhibidores de Proteínas Quinasas/farmacología , Animales , Proteínas de Ciclo Celular/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Xenoinjertos , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Ratones , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Secuencias Repetidas en Tándem , Tirosina Quinasa 3 Similar a fms/genéticaRESUMEN
We recently discovered that the protein phosphatase 2A (PP2A) B55α subunit (PPP2R2A) is under-expressed in primary blast cells and is unfavorable for remission duration in AML patients. In this study, reverse phase protein analysis (RPPA) of 230 proteins in 511 AML patient samples revealed a strong correlation of B55α with a number of proteins including MYC, PKC α, and SRC. B55α suppression in OCI-AML3 cells by shRNA demonstrated that the B subunit is a PKCα phosphatase. B55α does not target SRC, but rather the kinase suppresses protein expression of the B subunit. Finally, the correlation between B55α and MYC levels reflected a complex stoichiometric competition between B subunits. Loss of B55α in OCI-AML3 cells did not change global PP2A activity and the only isoform that is induced is the one containing B56α. In cells containing B55α shRNA, MYC was suppressed with concomitant induction of the competing B subunit B56α (PPP2R5A). A recent study determined that FTY-720, a drug whose action involves the activation of PP2A, resulted in the induction of B55α In AML cells, and a reduction of the B subunit rendered these cells resistant to FTY-720. Finally, reduction of the B subunit resulted in an increase in the expression of miR-191-5p and a suppression of miR-142-3p. B55α regulation of these miRs was intriguing as high levels of miR-191 portend poor survival in AML, and miR-142-3p is mutated in 2% of AML patient samples. In summary, the suppression of B55α activates signaling pathways that could support leukemia cell survival.
Asunto(s)
Leucemia Mieloide Aguda/enzimología , Leucemia Mieloide Aguda/genética , MicroARNs/genética , Proteína Fosfatasa 2/metabolismo , Transducción de Señal/genética , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Clorhidrato de Fingolimod , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Humanos , MicroARNs/metabolismo , Modelos Biológicos , Fosforilación/efectos de los fármacos , Glicoles de Propileno/farmacología , Proteína Quinasa C-alfa/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , ARN Interferente Pequeño/metabolismo , Transducción de Señal/efectos de los fármacos , Esfingosina/análogos & derivados , Esfingosina/farmacología , Familia-src Quinasas/metabolismoRESUMEN
BCL-2 proteins are critical for cell survival and are overexpressed in many tumors. ABT-737 is a small-molecule BH3 mimetic that exhibits single-agent activity against lymphoma and small-cell lung cancer in preclinical studies. We here report that ABT-737 effectively kills acute myeloid leukemia blast, progenitor, and stem cells without affecting normal hematopoietic cells. ABT-737 induced the disruption of the BCL-2/BAX complex and BAK-dependent but BIM-independent activation of the intrinsic apoptotic pathway. In cells with phosphorylated BCL-2 or increased MCL-1, ABT-737 was inactive. Inhibition of BCL-2 phosphorylation and reduction of MCL-1 expression restored sensitivity to ABT-737. These data suggest that ABT-737 could be a highly effective antileukemia agent when the mechanisms of resistance identified here are considered.
Asunto(s)
Apoptosis/fisiología , Compuestos de Bifenilo , Resistencia a Antineoplásicos/fisiología , Leucemia Mieloide Aguda , Nitrofenoles , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Sulfonamidas , Animales , Compuestos de Bifenilo/metabolismo , Compuestos de Bifenilo/uso terapéutico , Línea Celular , Dimerización , Células Madre Hematopoyéticas/fisiología , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/metabolismo , Ratones , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Proteínas de Neoplasias/metabolismo , Nitrofenoles/metabolismo , Nitrofenoles/uso terapéutico , Piperazinas/metabolismo , Piperazinas/uso terapéutico , Conformación Proteica , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-bcl-2/química , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Sulfonamidas/metabolismo , Sulfonamidas/uso terapéutico , Proteína Destructora del Antagonista Homólogo bcl-2/genética , Proteína Destructora del Antagonista Homólogo bcl-2/metabolismo , Proteína X Asociada a bcl-2/química , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Platelets promote angiogenesis by releasing angiogenesis-regulating factors from their α-granules upon aggregation. This effect has both physiologic and pathologic significance as it may contribute to carcinogenesis. Platelet α-granule release and aggregation are regulated, in part, via protein kinase C (PKC) α and ß signaling. Our study investigated the effects of PKC inhibition on aggregation, angiogenesis-regulator secretion from α-granules, and platelet-stimulated angiogenesis. We hypothesized that selective PKCα inhibition may preferentially suppress angiogenesis-regulator secretion from α-granules but not aggregation, limiting platelet-stimulated angiogenesis. Human platelets were aggregated in the presence of conventional PKC inhibitors myr-FARKGALRQ and Ro 32-0432 (2-{8-[(dimethylamino)methyl]-6,7,8,9-tetrahydropyridol[1,2-α]indol-3-yl}-3-(1-methyl-1H-indol-3-yl)maleimide). Immunofluorescence microscopy of PKC translocation was used to determine the specificity of PKC-inhibitor targeting. Enzyme-linked immunosorbent assay was used to measure vascular endothelial growth factor (VEGF) and thrombospondin-1 (TSP-1) release from platelets. Platelet effects on angiogenesis were tested using a capillary-formation assay. Ro 32-0432, but not the peptide inhibitor myr-FARKGALRQ (myristoylated-pseudosubstrate peptide inhibitor), inhibited aggregation in a concentration-dependent manner, while both Ro 32-0432 and myr-FARKGALRQ preferentially suppressed VEGF over TSP-1 secretion. Suppression of angiogenesis-regulator release occurred at inhibitor concentrations that did not significantly affect aggregation. Immunofluorescence microscopy revealed that PKCα targeting to α-granules is inhibited when angiogenesis-regulator secretion is uncoupled from aggregation. At concentrations that uncoupled α-granule release from aggregation, Ro 32-0432 and myr-FARKGALRQ inhibited platelet-stimulated angiogenesis. Hence, selective PKCα inhibition suppresses angiogenesis-regulator release from platelet α-granules with minimal effects on aggregation. Thus, selective PKCα inhibitors may have pharmacologic significance to regulate platelet-promoted angiogenesis.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Plaquetas/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Neovascularización Fisiológica/efectos de los fármacos , Agregación Plaquetaria/efectos de los fármacos , Proteína Quinasa C-alfa/antagonistas & inhibidores , Plaquetas/enzimología , Western Blotting , Técnicas de Cultivo de Célula , Células Cultivadas , Colágeno/farmacología , Gránulos Citoplasmáticos/efectos de los fármacos , Gránulos Citoplasmáticos/enzimología , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Humanos , Microscopía Fluorescente , Trombospondina 1/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Glycolipid transfer protein (GLTP) accelerates glycolipid intermembrane transfer via a unique lipid transfer/binding fold (GLTP fold) that defines the GLTP superfamily and is the prototype for functional GLTP-like domains in larger proteins, i.e. FAPP2. Human GLTP is encoded by the single-copy GLTP gene on chromosome 12 (12q24.11 locus), but regulation of GLTP gene expression remains completely unexplored. Herein, the ability of glycosphingolipids (and their sphingolipid metabolites) to regulate the transcriptional expression of GLTP via its promoter has been evaluated. Using luciferase and GFP reporters in concert with deletion mutants, the constitutive and basal (225 bp; â¼78% G+C) human GLTP promoters have been defined along with adjacent regulatory elements. Despite high G+C content, translational regulation was not evident by the mammalian target of rapamycin pathway. Four GC-boxes were shown to be functional Sp1/Sp3 transcription factor binding sites. Mutation of one GC-box was particularly detrimental to GLTP transcriptional activity. Sp1/Sp3 RNA silencing and mithramycin A treatment significantly inhibited GLTP promoter activity. Among tested sphingolipid analogs of glucosylceramide, sulfatide, ganglioside GM1, ceramide 1-phosphate, sphingosine 1-phosphate, dihydroceramide, sphingosine, only ceramide, a nonglycosylated precursor metabolite unable to bind to GLTP protein, induced GLTP promoter activity and raised transcript levels in vivo. Ceramide treatment partially blocked promoter activity decreases induced by Sp1/Sp3 knockdown. Ceramide treatment also altered the in vivo binding affinity of Sp1 and Sp3 for the GLTP promoter and decreased Sp3 acetylation. This study represents the first characterization of any Gltp gene promoter and links human GLTP expression to sphingolipid homeostasis through ceramide.
Asunto(s)
Proteínas Portadoras/genética , Ceramidas/metabolismo , Factor de Transcripción Sp1/metabolismo , Factor de Transcripción Sp3/metabolismo , Transcripción Genética/fisiología , Secuencia de Aminoácidos , Secuencia de Bases , Neoplasias de la Mama , Femenino , Silenciador del Gen , Células HEK293 , Células HeLa , Humanos , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/fisiología , Transducción de Señal/fisiología , Serina-Treonina Quinasas TOR/metabolismo , Sitio de Iniciación de la Transcripción/fisiología , Regulación hacia Arriba/fisiologíaRESUMEN
The chronic lymphocytic leukemia (CLL) armamentarium has evolved significantly, with novel therapies that inhibit Bruton Tyrosine Kinase, PI3K delta and/or the BCL2 protein improving outcomes. Still, the clinical course of CLL patients is highly variable and most previously recognized prognostic features lack the capacity to predict response to modern treatments indicating the need for new prognostic markers. In this study, we identified four epigenetically distinct proteomic signatures of a large cohort of CLL and related diseases derived samples (n = 871) using reverse phase protein array technology. These signatures are associated with clinical features including age, cytogenetic abnormalities [trisomy 12, del(13q) and del(17p)], immunoglobulin heavy-chain locus (IGHV) mutational load, ZAP-70 status, Binet and Rai staging as well as with the outcome measures of time to treatment and overall survival. Protein signature membership was identified as predictive marker for overall survival regardless of other clinical features. Among the analyzed epigenetic proteins, EZH2, HDAC6, and loss of H3K27me3 levels were the most independently associated with poor survival. These findings demonstrate that proteomic based epigenetic biomarkers can be used to better classify CLL patients and provide therapeutic guidance.
Asunto(s)
Epigénesis Genética , Regulación Leucémica de la Expresión Génica , Leucemia Linfocítica Crónica de Células B/genética , Anciano , Aberraciones Cromosómicas , Femenino , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Masculino , Persona de Mediana Edad , Mutación , ProteómicaRESUMEN
Recent studies in acute myeloid leukemia (AML) suggest activation of pro-proliferative signaling cascades including those mediated by protein kinase C (PKC) represent a poor prognostic factor for patients. The classical PKC isoforms α and ß generally support survival signaling and have emerged as important targets for anti-cancer therapy. Enzastaurin is a PKC ß inhibitor and is in clinical trials for lymphomas, gliomas, and lung cancer. Presently, it is not known if enzastaurin could be effective against AML. In the current study, we found that high dose enzastaurin was found to promote apoptosis in the AML-derived cell lines and in blast cells from AML patients. The mechanism of cell death, however, likely does not involve PKC ß as another PKC ß inhibitor was not toxic to AML cell lines and did not promote enzastaurin-induced cell killing. While enzastaurin is fairly specific for PKC ß, the agent can inhibit other PKC isoforms at higher concentrations. Enzastaurin was effective at inhibiting PKC α phosphorylation and membrane localization in the AML cell lines and suppressed phosphorylation of BCL2. Furthermore, enzastaurin suppressed activation of ERK (which can be activated by PKC α). Analysis of the serine/threonine phosphorylation profile in HL60 cells after enzastaurin treatment revealed that the drug inhibits the phosphorylation of a distinct set of proteins while promoting phosphorylation of another set of proteins. This suggests the drug may regulate multiple signaling pathways. Taken together, these findings suggest that enzastaurin could be effective in the therapy of AML.
Asunto(s)
Apoptosis/efectos de los fármacos , Indoles/farmacología , Leucemia Mieloide Aguda/metabolismo , Proteína Quinasa C/metabolismo , Antígenos CD34/metabolismo , Western Blotting , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Electroforesis en Gel Bidimensional , Células HL-60 , Humanos , Microscopía Fluorescente , Fosforilación/efectos de los fármacos , Proteína Quinasa C beta , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genéticaRESUMEN
Receptor Tyrosine Kinases are critical regulators of signal transduction that support cell survival, proliferation, and differentiation. Dysregulation of normal Receptor Tyrosine Kinase function by mutation or other activity-altering event can be oncogenic or can impact the transformed malignant cell so it becomes particularly resistant to stress challenge, have increased proliferation, become evasive to immune surveillance, and may be more prone to metastasis of the tumor to other organ sites. The TAM family of Receptor Tyrosine Kinases (TYRO3, AXL, MERTK) is emerging as important components of malignant cell survival in many cancers. The TAM kinases are important regulators of cellular homeostasis and proper cell differentiation in normal cells as receptors for their ligands GAS6 and Protein S. They also are critical to immune and inflammatory processes. In malignant cells, the TAM kinases can act as ligand independent co-receptors to mutant Receptor Tyrosine Kinases and in some cases (e.g. FLT3-ITD mutant) are required for their function. They also have a role in immune checkpoint surveillance. At the time of this review, the Covid-19 pandemic poses a global threat to world health. TAM kinases play an important role in host response to many viruses and it is suggested the TAM kinases may be important in aspects of Covid-19 biology. This review will cover the TAM kinases and their role in these processes.
Asunto(s)
Muerte Celular , Inmunidad , Proteínas Tirosina Quinasas Receptoras/inmunología , Virosis/inmunología , Animales , COVID-19/genética , COVID-19/inmunología , COVID-19/metabolismo , Humanos , Inflamación/genética , Inflamación/inmunología , Inflamación/metabolismo , Mutación , Neoplasias/genética , Neoplasias/inmunología , Neoplasias/metabolismo , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Virosis/genética , Virosis/metabolismoRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is a disease of aging. The TP53 gene product regulates cell growth, aging, and cancer. To determine the important targets of TP53 in PDAC, we examined the expression of 440 proteins on a reverse phase protein array (RPPA) in PDAC-derived MIA-PaCa-2 cells which either had WT-TP53 or lacked WT-TP53. MIA-PaCa-2 cells have a TP53 mutation as well as mutant KRAS and represent a good in vitro model to study PDAC. RPPA analysis demonstrated expression of tumor promoting proteins in cells that lacked WT-TP53; and this feature could be reversed significantly when the cells were transfected with vector encoding WT-TP53 or treated with berberine or a modified berberine (BBR). Expression of miR-34a-associated signaling was elevated in cells expressing WT-TP53 compared to cells expressing mTP53. Results from in vivo studies using human PDAC specimens confirmed the in vitro results as the expression of miR-34a and associated signaling was significantly decreased in PDAC specimens compared to non-cancerous tissues. This study determined SERPINE1 as a miR-34a target with relevance to the biology of PDAC. Thus, we have identified a key target (SERPINE1) of the TP53/miR-34a axis that may serve as a potential biomarker for early detection of pancreatic cancer.
Asunto(s)
Regulación Neoplásica de la Expresión Génica/fisiología , MicroARNs/metabolismo , Neoplasias Pancreáticas/metabolismo , Inhibidor 1 de Activador Plasminogénico/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Antineoplásicos/farmacología , Apoptosis , Berberina/farmacología , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Supervivencia Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , MicroARNs/genética , Inhibidor 1 de Activador Plasminogénico/genética , Transducción de Señal , Proteína p53 Supresora de Tumor/genéticaRESUMEN
The galectin LGALS1 is a glycan binding protein that regulates intracellular (e.g. signal transduction) and extracellular processes (e.g. immunity, leukocyte mobilization) that support cell survival. The protein is best known for its role in RAS signaling. LGALS1 is important in acute lymphoblastic leukemia but its role in acute myeloid leukemia is not well defined. We previously found suppression of LGALS1 in AML cell lines OCI-AML3 and THP-1 sensitized both cell lines to BCL2 inhibitor ABT-737. In this study, we used an in vivo murine OCI-AML3 xenograft model to test whether reduction expression of LGALS1 affects survival. Mice bearing the OCI-AML3 cells with LGALS1 shRNA survived significantly longer than mice with control OCI-AML3 cells. Gene expression profiling using RNASeq was performed using the control and LGALS1 shRNA of p53 WT OCI-AML3 and p53 mutant THP-1 cells. The data reveal distinct differences between the two cell lines in number of genes affected, in pathways associated with these genes, in expression of oncogenes, and in the transcription factors involved. The p53 pathway is prominent in OCI-AML3 cells. An examination of LGALS1 mRNA in an AML patient population reveals elevated LGALS1 mRNA is associated with shorter disease free survival and increased blasts in the BM. This data with the xenograft model data presented suggest LGALS1 may be important in the AML microenvironment. In summary, the data presented here suggest that a strategy targeting LGALS1 may benefit AML patients.
Asunto(s)
Galectina 1/metabolismo , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Animales , Médula Ósea/patología , Línea Celular Tumoral , Supervivencia Celular , Galectina 1/genética , Regulación Leucémica de la Expresión Génica , Ontología de Genes , Humanos , Leucemia Mieloide Aguda/genética , Masculino , Ratones Endogámicos NOD , Ratones SCID , Pronóstico , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Bazo/patología , Células THP-1 , Carga Tumoral , Proteína p53 Supresora de Tumor/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Cancer cells depend on a supportive niche (the tumor microenvironment) that promotes tumor cell survival while protecting the malignant cells from therapeutic challenges and the host's defense systems. Cancer cells and the support cells in the tumor microenvironment communicate via cytokines/chemokines, cell:cell contact, or alterations in the metabolic state of the niche (e.g. hypoxia) that promote growth and survival of the tumor cell, influence metastasis, and defeat immune surveillance. These signaling pathways involve dysregulation of not only protein kinases but also protein phosphatases as normal signal transduction processes require both activation and deactivation. For instance, aberrant receptor signaling can result from constitutive activation of a tyrosine kinase such as FLT3 or inactivation of a tyrosine protein phosphatase such as SHP-2 (PTPN11). Activation of serine/threonine kinases such as AKT and ERK are often observed during the development of drug resistance while genomic and non-genomic suppression of serine/threonine protein phosphatases such as PP2A achieve similar results. It is fairly clear that the various protein phosphatases will impact processes that support drug resistance. Of growing interest is the emerging model whereby the support cells in the tumor microenvironment actually serve as drivers of tumorigenesis. This phenomenon has been most prominently observed in osteoblast cells in leukemic niches. At least one protein phosphatase, PTPN11, has emerged as a critical driver of this process in juvenile myelomonocytic leukemia. This review will cover the role of various serine/threonine and tyrosine protein phosphatases in processes that are central to tumor microenvironment function.