RESUMEN
Although highly effective, BCR-ABL1 tyrosine kinase inhibitors do not target chronic myeloid leukemia (CML) stem cells. Most patients relapse upon tyrosine kinase inhibitor therapy cessation. We reported previously that combined BCR-ABL1 and BCL-2 inhibition synergistically targets CML stem/progenitor cells. p53 induces apoptosis mainly by modulating BCL-2 family proteins. Although infrequently mutated in CML, p53 is antagonized by MDM2, which is regulated by BCR-ABL1 signaling. We hypothesized that MDM2 inhibition could sensitize CML cells to tyrosine kinase inhibitors. Using an inducible transgenic Scl-tTa-BCR-ABL1 murine CML model, we found, by RT-PCR and CyTOF proteomics increased p53 signaling in CML bone marrow (BM) cells compared with controls in CD45+ and linage-SCA-1+C-KIT+ populations. CML BM cells were more sensitive to exogenous BH3 peptides than controls. Combined inhibition of BCR-ABL1 with imatinib and MDM2 with DS-5272 increased NOXA level, markedly reduced leukemic linage-SCA-1+C-KIT+ cells and hematopoiesis, decreased leukemia burden, significantly prolonged the survival of mice engrafted with BM cells from Scl-tTa-BCR-ABL1 mice, and significantly decreased CML stem cell frequency in secondary transplantations. Our results suggest that CML stem/progenitor cells have increased p53 signaling and a propensity for apoptosis. Combined MDM2 and BCR-ABL1 inhibition targets CML stem/progenitor cells and has the potential to improve cure rates for CML.
Asunto(s)
Leucemia Mielógena Crónica BCR-ABL Positiva , Inhibidores de Proteínas Quinasas , Animales , Proliferación Celular , Modelos Animales de Enfermedad , Resistencia a Antineoplásicos , Proteínas de Fusión bcr-abl/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Ratones , Células Madre Neoplásicas , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-mdm2/genéticaRESUMEN
The pathogenesis of acute myeloid leukemia (AML) involves serial acquisition of mutations controlling several cellular processes, requiring combination therapies affecting key downstream survival nodes in order to treat the disease effectively. The BCL2 selective inhibitor venetoclax has potent anti-leukemia efficacy; however, resistance can occur due to its inability to inhibit MCL1, which is stabilized by the MAPK pathway. In this study, we aimed to determine the anti-leukemia efficacy of concomitant targeting of the BCL2 and MAPK pathways by venetoclax and the MEK1/2 inhibitor cobimetinib, respectively. The combination demonstrated synergy in seven of 11 AML cell lines, including those resistant to single agents, and showed growth-inhibitory activity in over 60% of primary samples from patients with diverse genetic alterations. The combination markedly impaired leukemia progenitor functions, while maintaining normal progenitors. Mass cytometry data revealed that BCL2 protein is enriched in leukemia stem/progenitor cells, primarily in venetoclax-sensitive samples, and that cobimetinib suppressed cytokine-induced pERK and pS6 signaling pathways. Through proteomic profiling studies, we identified several pathways inhibited downstream of MAPK that contribute to the synergy of the combination. In OCI-AML3 cells, the combination downregulated MCL1 protein levels and disrupted both BCL2:BIM and MCL1:BIM complexes, releasing BIM to induce cell death. RNA sequencing identified several enriched pathways, including MYC, mTORC1, and p53 in cells sensitive to the drug combination. In vivo, the venetoclax-cobimetinib combination reduced leukemia burden in xenograft models using genetically engineered OCI-AML3 and MOLM13 cells. Our data thus provide a rationale for combinatorial blockade of MEK and BCL2 pathways in AML.
Asunto(s)
Leucemia Mieloide Aguda , Proteómica , Apoptosis , Azetidinas , Compuestos Bicíclicos Heterocíclicos con Puentes , Línea Celular Tumoral , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Piperidinas , Proteínas Proto-Oncogénicas c-bcl-2/genética , SulfonamidasRESUMEN
Understanding the unique phenotypes and complex signaling pathways of leukemia stem cells (LSCs) will provide insights and druggable targets that can be used to eradicate acute myeloid leukemia (AML). Current work on AML LSCs is limited by the number of parameters that conventional flow cytometry (FCM) can analyze because of cell autofluorescence and fluorescent dye spectral overlap. Single-cell mass cytometry (CyTOF) substitutes rare earth elements for fluorophores to label antibodies, which allows measurements of up to 120 parameters in single cells without correction for spectral overlap. The aim of this study was the evaluation of intracellular signaling in antigen-defined stem/progenitor cell subsets in primary AML. CyTOF and conventional FCM yielded comparable results on LSC phenotypes defined by CD45, CD34, CD38, CD123, and CD99. Intracellular phosphoprotein responses to ex vivo cell signaling inhibitors and cytokine stimulation were assessed in myeloid leukemia cell lines and one primary AML sample. CyTOF and conventional FCM results were confirmed by western blotting. In the primary AML sample, we investigated the cell responses to ex vivo stimulation with stem cell factor and BEZ235-induced inhibition of PI3K and identified activation patterns in multiple PI3K downstream signaling pathways including p-4EBP1, p-AKT, and p-S6, particularly in CD34(+) subsets. We evaluated multiple signaling pathways in antigen-defined subpopulations in primary AML cells with FLT3-ITD mutations. The data demonstrated the heterogeneity of cell phenotype distribution and distinct patterns of signaling activation across AML samples and between AML and normal samples. The mTOR targets p-4EBP1 and p-S6 were exclusively found in FLT3-ITD stem/progenitor cells, but not in their normal counterparts, suggesting both as novel targets in FLT3 mutated AML. Our data suggest that CyTOF can identify functional signaling pathways in antigen-defined subpopulations in primary AML, which may provide a rationale for designing therapeutics targeting LSC-enriched cell populations.
Asunto(s)
Citometría de Flujo/métodos , Leucemia Mieloide Aguda/genética , Células Madre Neoplásicas/citología , Transducción de Señal/genética , Tirosina Quinasa 3 Similar a fms/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Antígenos CD/genética , Antígenos CD/inmunología , Proteínas de Ciclo Celular , Línea Celular Tumoral , Proliferación Celular/genética , Supervivencia Celular/genética , Citocinas/metabolismo , Humanos , Imidazoles/farmacología , Espectrometría de Masas/métodos , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Quinolinas/farmacología , Proteínas Quinasas S6 Ribosómicas/metabolismo , Coloración y Etiquetado , Factor de Células Madre/farmacología , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Overexpression of the apoptosis repressor with caspase recruitment domain (ARC, also termed NOL3) protein predicts adverse outcome in patients with acute myeloid leukaemia (AML) and confers drug resistance to AML cells. The second mitochondrial-derived activator of caspases (SMAC, also termed DIABLO) mimetic, birinapant, promotes extrinsic apoptosis in AML cells. SMAC mimetics induce cleavage of cellular inhibitor of apoptosis (cIAP) proteins, leading to stabilization of the nuclear factor-κB (NF-κB)-inducing kinase (MAP3K14, also termed NIK) and activation of non-canonical NF-κB signalling. To enhance the therapeutic potential of SMAC mimetics in AML, we investigated the regulation and role of ARC in birinapant-induced apoptosis. We showed that birinapant increases ARC in AML and bone marrow-derived mesenchymal stromal cells (MSCs). Downregulation of MAP3K14 by siRNA decreased ARC levels and suppressed birinapant-induced ARC increase. Reverse-phase protein array analysis of 511 samples from newly diagnosed AML patients showed that BIRC2 (also termed cIAP1) and ARC were inversely correlated. Knockdown of ARC sensitized, while overexpression attenuated, birinapant-induced apoptosis. Furthermore, ARC knockdown in MSCs sensitized co-cultured AML cells to birinapant-induced apoptosis. Our data demonstrate that ARC is regulated via BIRC2/MAP3K14 signalling and its overexpression in AML or MSCs can function as a resistant factor to birinapant-induced leukaemia cell death, suggesting that strategies to inhibit ARC will improve the therapeutic potential of SMAC mimetics.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/fisiología , Apoptosis/fisiología , Dipéptidos/farmacología , Regulación Leucémica de la Expresión Génica , Indoles/farmacología , Proteínas Inhibidoras de la Apoptosis/fisiología , Péptidos y Proteínas de Señalización Intracelular/fisiología , Leucemia Mieloide Aguda/patología , Sistema de Señalización de MAP Quinasas/fisiología , Células Madre Mesenquimatosas/efectos de los fármacos , Proteínas Mitocondriales/fisiología , Proteínas Musculares/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Anciano , Antimetabolitos Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/antagonistas & inhibidores , Proteínas Reguladoras de la Apoptosis/genética , Técnicas de Cocultivo , Dipéptidos/uso terapéutico , Diseño de Fármacos , Resistencia a Antineoplásicos , Humanos , Indoles/uso terapéutico , Proteínas Inhibidoras de la Apoptosis/genética , Leucemia Mieloide Aguda/sangre , Leucemia Mieloide Aguda/genética , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Persona de Mediana Edad , Terapia Molecular Dirigida , Proteínas Musculares/antagonistas & inhibidores , Proteínas Musculares/genética , FN-kappa B/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Interferencia de ARN , ARN Interferente Pequeño/farmacología , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/farmacología , Ubiquitina-Proteína Ligasas , Quinasa de Factor Nuclear kappa BRESUMEN
Survivin, a member of the inhibitors of apoptosis protein family, plays important roles in cell proliferation and survival and is highly expressed in various malignancies, including leukemias. To better understand its role in acute myeloid leukemia (AML), we profiled survivin expression in samples obtained from 511 newly diagnosed AML patients and in CD34(+)38(-) AML stem/progenitor cells using a validated reverse-phase protein array; we correlated its levels with clinical outcomes and with levels of other proteins in the same sample set. We found that survivin levels were higher in bone marrow than in paired peripheral blood leukemic cells (n = 140, P = .0001) and that higher survivin levels significantly predicted shorter overall (P = .016) and event-free (P = .023) survival in multivariate Cox model analysis. Importantly, survivin levels were significantly higher in CD34(+)38(-) AML stem/progenitor cells than in bulk blasts and total CD34(+) AML cells (P < .05). Survivin expression correlated with the expressions of multiple proteins involved with cell proliferation and survival. Particularly, its expression strongly correlated with HIF1α in the stem/progenitor cell compartment. These results suggest that survivin is a prognostic biomarker in AML and that survivin, which is overexpressed in AML stem/progenitor cells, remains a potentially important target for leukemia therapy.
Asunto(s)
Antineoplásicos/uso terapéutico , Células Madre Hematopoyéticas/fisiología , Proteínas Inhibidoras de la Apoptosis/metabolismo , Leucemia Mieloide Aguda , ADP-Ribosil Ciclasa 1/metabolismo , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Antígenos CD34/metabolismo , Biomarcadores de Tumor/metabolismo , Proliferación Celular , Supervivencia Celular/fisiología , Resistencia a Antineoplásicos/fisiología , Femenino , Células Madre Hematopoyéticas/patología , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/mortalidad , Masculino , Glicoproteínas de Membrana/metabolismo , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Análisis por Matrices de Proteínas , Análisis de Supervivencia , Survivin , Adulto JovenRESUMEN
Regulators of apoptosis in acute myeloid leukemia (AML) have been extensively studied and are considered excellent therapeutic targets. Apoptosis repressor with caspase recruitment domain (ARC), an antiapoptotic protein originally found to be involved in apoptosis of cardiac cells, was recently demonstrated to be overexpressed in several solid tumors. To assess its importance in AML, we profiled ARC expression in 511 newly diagnosed AML patients using a validated robust reverse-phase protein array and correlated ARC levels with clinical outcomes. ARC was variably expressed in samples from patients with AML. ARC level was not associated with cytogenetic groups or with FLT-3 mutation status. However, patients with low or medium ARC protein levels had significantly better outcomes than those with high ARC levels: longer overall survival (median, 53.9 or 61.6 vs 38.9 weeks, P = .0015) and longer remission duration (median, 97.6 or 44.7 vs 31.1 weeks, P = .0007). Multivariate analysis indicated that ARC was a statistically significant independent predictor of survival in AML (P = .00013). Inhibition of ARC promoted apoptosis and sensitized cytosine arabinoside-induced apoptosis in OCI-AML3 cells. These results suggest that ARC expression levels are highly prognostic in AML and that ARC is a potential therapeutic target in AML.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Leucemia Mieloide/metabolismo , Proteínas Musculares/metabolismo , Enfermedad Aguda , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/genética , Western Blotting , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Citarabina/farmacología , Femenino , Humanos , Leucemia Mieloide/diagnóstico , Leucemia Mieloide/genética , Masculino , Persona de Mediana Edad , Análisis Multivariante , Proteínas Musculares/genética , Mutación , Oligonucleótidos Antisentido/genética , Pronóstico , Análisis de Supervivencia , Adulto Joven , Tirosina Quinasa 3 Similar a fms/genéticaRESUMEN
Checkpoint immunotherapy has yielded meaningful responses across many cancers but has shown modest efficacy in advanced prostate cancer. B7 homolog 3 protein (B7-H3/CD276) is an immune checkpoint molecule and has emerged as a promising therapeutic target. However, much remains to be understood regarding B7-H3's role in cancer progression, predictive biomarkers for B7-H3-targeted therapy, and combinatorial strategies. Our multi-omics analyses identified B7-H3 as one of the most abundant immune checkpoints in prostate tumors containing PTEN and TP53 genetic inactivation. Here, we sought in vivo genetic evidence for, and mechanistic understanding of, the role of B7-H3 in PTEN/TP53-deficient prostate cancer. We found that loss of PTEN and TP53 induced B7-H3 expression by activating transcriptional factor Sp1. Prostate-specific deletion of Cd276 resulted in delayed tumor progression and reversed the suppression of tumor-infiltrating T cells and NK cells in Pten/Trp53 genetically engineered mouse models. Furthermore, we tested the efficacy of the B7-H3 inhibitor in preclinical models of castration-resistant prostate cancer (CRPC). We demonstrated that enriched regulatory T cells and elevated programmed cell death ligand 1 (PD-L1) in myeloid cells hinder the therapeutic efficacy of B7-H3 inhibition in prostate tumors. Last, we showed that B7-H3 inhibition combined with blockade of PD-L1 or cytotoxic T lymphocyte-associated protein 4 (CTLA-4) achieved durable antitumor effects and had curative potential in a PTEN/TP53-deficient CRPC model. Given that B7-H3-targeted therapies have been evaluated in early clinical trials, our studies provide insights into the potential of biomarker-driven combinatorial immunotherapy targeting B7-H3 in prostate cancer, among other malignancies.
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Neoplasias de la Próstata , Humanos , Masculino , Animales , Ratones , Línea Celular Tumoral , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Factor de Transcripción Sp1/metabolismo , Regulación hacia Arriba , Progresión de la EnfermedadRESUMEN
Activation of p53 by murine double minute (MDM2) antagonist nutlin-3a or inhibition of X-linked inhibitor of apoptosis (XIAP) induces apoptosis in acute myeloid leukemia (AML) cells. We demonstrate that concomitant inhibition of MDM2 by nutlin-3a and of XIAP by small molecule antagonists synergistically induced apoptosis in p53 wild-type OCI-AML3 and Molm13 cells. Knockdown of p53 by shRNA blunted the synergy, and down-regulation of XIAP by antisense oligonucleotide (ASO) enhanced nutlin-3a-induced apoptosis, suggesting that the synergy was mediated by p53 activation and XIAP inhibition. This is supported by data showing that inhibition of both MDM2 and XIAP by their respective ASOs induced significantly more cell death than either ASO alone. Importantly, p53 activation and XIAP inhibition enhanced apoptosis in blasts from patients with primary AML, even when the cells were protected by stromal cells. Mechanistic studies demonstrated that XIAP inhibition potentiates p53-induced apoptosis by decreasing p53-induced p21 and that p53 activation enhances XIAP inhibition-induced cell death by promoting mitochondrial release of second mitochondria-derived activator of caspases (SMAC) and by inducing the expression of caspase-6. Because both XIAP and p53 are presently being targeted in ongoing clinical trials in leukemia, the combination strategy holds promise for expedited translation into the clinic.
Asunto(s)
Apoptosis , Crisis Blástica/metabolismo , Leucemia Mieloide Aguda/metabolismo , Transducción de Señal , Proteína p53 Supresora de Tumor/metabolismo , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis , Crisis Blástica/genética , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Caspasa 6/biosíntesis , Caspasa 6/genética , Línea Celular Tumoral , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/genética , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Regulación Leucémica de la Expresión Génica/genética , Técnicas de Silenciamiento del Gen , Humanos , Imidazoles/farmacología , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Leucemia Mieloide Aguda/genética , Ratones , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Piperazinas/farmacología , Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Células del Estroma/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína Inhibidora de la Apoptosis Ligada a X/genéticaRESUMEN
MCL-1 is known to play a major role in resistance to BCL-2 inhibition, but the contribution of other BCL-2 family proteins has not been fully explored. We, here, demonstrate the ineffectiveness of MCL-1 inhibitor AMG176 in venetoclax-resistant, and conversely, of venetoclax in AMG176-resistant acute myelogenous leukemia (AML). Like cells with acquired resistance to venetoclax, cells with acquired resistance to AMG176 express increased MCL-1. Both cells with acquired resistance to venetoclax and to AMG176 express increased levels of BCL-2 and BCL-2A1, decreased BAX, and/or altered levels of other BCL-2 proteins. Cotargeting BCL-2 and MCL-1 was highly synergistic in AML cell lines with intrinsic or acquired resistance to BH3 mimetics or engineered to genetically overexpress BCL-2 or BCL-2A1 or downregulate BAX. The combination effectively eliminated primary AML blasts and stem/progenitor cells resistant to or relapsed after venetoclax-based therapy irrespective of mutations and cytogenetic abnormalities. Venetoclax and AMG176 combination markedly suppressed antiapoptotic BCL-2 proteins and AML stem/progenitor cells and dramatically extended mouse survival (median 336 vs. control 126 days; P < 0.0001) in a patient-derived xenograft (PDX) model developed from a venetoclax/hypomethylating agent therapy-resistant patient with AML. However, decreased BAX levels in the bone marrow residual leukemia cells after 4-week combination treatment may represent a resistance mechanism that contributed to their survival. Enhanced antileukemia activity was also observed in a PDX model of monocytic AML, known to be resistant to venetoclax therapy. Our results support codependence on multiple antiapoptotic BCL-2 proteins and suppression of BAX as mechanisms of AML resistance to individual BH3 mimetics. Cotargeting of MCL-1 and BCL-2 eliminates otherwise apoptosis-resistant cells.
Asunto(s)
Proteínas Reguladoras de la Apoptosis , Materiales Biomiméticos , Leucemia Mieloide Aguda , Animales , Apoptosis , Materiales Biomiméticos/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Línea Celular Tumoral , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/metabolismo , Ratones , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , Proteínas Proto-Oncogénicas c-bcl-2 , Células Madre/metabolismo , Proteína X Asociada a bcl-2/metabolismo , Proteína X Asociada a bcl-2/farmacologíaRESUMEN
XIAP, a potent caspase inhibitor, is highly expressed in acute myeloid leukemia (AML) cells and contributes to chemoresistance. A multi-center phase 1/2 trial of XIAP antisense oligonucleotide AEG35156 in combination with idarubicin/cytarabine was conducted in 56 patients with relapsed/refractory AML. Herein we report the pharmacodynamic studies of the patients enrolled at M. D. Anderson Cancer Center. A total of 13 patients were enrolled in our institution: five in phase 1 (12-350 mg/m² AEG35156) and eight in phase 2 (350 mg/m² AEG35156) of the protocol. AEG35156 was administered on 3 consecutive days and then weekly up to a maximum of 35 days. Blood samples were collected from patients on days 1 through 5 and on day 28-35 post-chemotherapy for detection of XIAP levels and apoptosis. AEG35156 treatment led to dose-dependent decreases of XIAP mRNA levels (42-100% reduction in phase 2 patients). XIAP protein levels were reduced in all five samples measured. Apoptosis induction was detected in 1/4 phase 1 and 4/5 phase 2 patients. Importantly, apoptosis was most pronounced in CD34+38- AML stem cells and all phase 2 patients showing apoptosis induction in CD34+38- cells achieved response. We conclude that at 350 mg/m², AEG35156 is effective in knocking down XIAP in circulating blasts accompanied by the preferential induction of apoptosis in CD34+38- AML stem cells.
Asunto(s)
Antimetabolitos Antineoplásicos/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Apoptosis/efectos de los fármacos , Citarabina/administración & dosificación , Idarrubicina/administración & dosificación , Oligonucleótidos/administración & dosificación , Proteína Inhibidora de la Apoptosis Ligada a X/antagonistas & inhibidores , Anciano , Antígenos CD/análisis , Antígenos CD/efectos de los fármacos , Caspasas/metabolismo , Esquema de Medicación , Cálculo de Dosificación de Drogas , Femenino , Expresión Génica/efectos de los fármacos , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Masculino , Persona de Mediana Edad , Células Progenitoras Mieloides/citología , Células Progenitoras Mieloides/metabolismo , ARN Mensajero/biosíntesis , Recurrencia , Proteína Inhibidora de la Apoptosis Ligada a X/genéticaRESUMEN
In B-cell acute lymphoblastic leukemia (B-ALL), activation of Notch signaling leads to cell-cycle arrest and apoptosis. We aimed to harness knowledge acquired by understanding a mechanism of Notch-induced cell death to elucidate a therapeutically viable target in B-ALL. To this end, we identified that Notch activation suppresses Polo-like kinase 1 (PLK1) in a B-ALL-specific manner. We identified that PLK1 is expressed in all subsets of B-ALL and is highest in Philadelphia-like (Ph-like) ALL, a high-risk subtype of disease. We biochemically delineated a mechanism of Notch-induced PLK1 downregulation that elucidated stark regulation of p53 in this setting. Our findings identified a novel posttranslational cascade initiated by Notch in which CHFR was activated via PARP1-mediated PARylation, resulting in ubiquitination and degradation of PLK1. This led to hypophosphorylation of MDM2Ser260, culminating in p53 stabilization and upregulation of BAX. shRNA knockdown or pharmacologic inhibition of PLK1 using BI2536 or BI6727 (volasertib) in B-ALL cell lines and patient samples led to p53 stabilization and cell death. These effects were seen in primary human B-ALL samples in vitro and in patient-derived xenograft models in vivo These results highlight PLK1 as a viable therapeutic target in B-ALL. Efficacy of clinically relevant PLK1 inhibitors in B-ALL patient-derived xenograft mouse models suggests that use of these agents may be tailored as an additional therapeutic strategy in future clinical studies.
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Proteínas de Ciclo Celular/antagonistas & inhibidores , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamiento farmacológico , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Pteridinas/farmacología , Receptores Notch/metabolismo , Animales , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Línea Celular Tumoral , Humanos , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Oncogenes , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Interferencia de ARN , Ensayos Antitumor por Modelo de Xenoinjerto/métodos , Quinasa Tipo Polo 1RESUMEN
Purpose: Wnt/ß-catenin signaling is required for leukemic stem cell function. FLT3 mutations are frequently observed in acute myeloid leukemia (AML). Anomalous FLT3 signaling increases ß-catenin nuclear localization and transcriptional activity. FLT3 tyrosine kinase inhibitors (TKI) are used clinically to treat FLT3-mutated AML patients, but with limited efficacy. We investigated the antileukemia activity of combined Wnt/ß-catenin and FLT3 inhibition in FLT3-mutant AML.Experimental Design: Wnt/ß-catenin signaling was inhibited by the ß-catenin/CBP antagonist C-82/PRI-724 or siRNAs, and FLT3 signaling by sorafenib or quizartinib. Treatments on apoptosis, cell growth, and cell signaling were assessed in cell lines, patient samples, and in vivo in immunodeficient mice by flow cytometry, Western blot, RT-PCR, and CyTOF.Results: We found significantly higher ß-catenin expression in cytogenetically unfavorable and relapsed AML patient samples and in the bone marrow-resident leukemic cells compared with circulating blasts. Disrupting Wnt/ß-catenin signaling suppressed AML cell growth, induced apoptosis, abrogated stromal protection, and synergized with TKIs in FLT3-mutated AML cells and stem/progenitor cells in vitro The aforementioned combinatorial treatment improved survival of AML-xenografted mice in two in vivo models and impaired leukemia cell engraftment. Mechanistically, the combined inhibition of Wnt/ß-catenin and FLT3 cooperatively decreased nuclear ß-catenin and the levels of c-Myc and other Wnt/ß-catenin and FLT3 signaling proteins. Importantly, ß-catenin inhibition abrogated the microenvironmental protection afforded the leukemic stem/progenitor cells.Conclusions: Disrupting Wnt/ß-catenin signaling exerts potent activities against AML stem/progenitor cells and synergizes with FLT3 inhibition in FLT3-mutant AML. These findings provide a rationale for clinical development of this strategy for treating FLT3-mutated AML patients. Clin Cancer Res; 24(10); 2417-29. ©2018 AACR.
Asunto(s)
Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Mutación , Vía de Señalización Wnt/efectos de los fármacos , Tirosina Quinasa 3 Similar a fms/genética , Animales , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Biomarcadores de Tumor , Ciclo Celular/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Sinergismo Farmacológico , Femenino , Silenciador del Gen , Humanos , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/tratamiento farmacológico , Ratones , Células Madre Neoplásicas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Transporte de Proteínas/efectos de los fármacos , Microambiente Tumoral/genética , Ensayos Antitumor por Modelo de Xenoinjerto , Tirosina Quinasa 3 Similar a fms/antagonistas & inhibidoresRESUMEN
Tetra-O-methyl nordihydroguaiaretic acid (M4N) was shown to induce G2 arrest and suppress human xenograft tumor growth by inhibiting Cdc2 and survivin. We examined the effect of M4N on leukemia and found that M4N inhibited growth and induced cell death in leukemic cell lines and blasts from AML patients. However, no significant changes in Cdc2 and survivin levels and G2 arrest were observed. Cell death and growth inhibition were dependent neither on XIAP, Bcl-2, and Bcl-X(L) levels nor on caspase-8. M4N did not promote cell differentiation in HL-60 cells. Interestingly, significant inhibition of AKT phosphorylation was observed in M4N treated OCI-AML3 cells. Collectively, our data showed that M4N inhibited cell growth and induced cell death in both leukemic cell lines and AML patient sample via a mechanism not mediated by Cdc2 and survivin inhibition and suggested that the extrinsic and the mitochondrial apoptotic pathways are not essential.
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Antioxidantes/farmacología , Proteína Quinasa CDC2/metabolismo , Leucemia/tratamiento farmacológico , Masoprocol/farmacología , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas de Neoplasias/metabolismo , Apoptosis , Ciclo Celular , Línea Celular Tumoral , Supervivencia Celular , Ensayos de Selección de Medicamentos Antitumorales , Células HL-60 , Humanos , Proteínas Inhibidoras de la Apoptosis , Células Jurkat , Mitocondrias/metabolismo , Survivin , Células U937RESUMEN
Mass cytometry presents an exceptional opportunity to interrogate the biology of highly heterogeneous cell populations, owing to the ability to collect highly parametric proteomic data at a single cell level. However, sample-to-sample variability, due to antibody staining and/or instrument sensitivity, can introduce substantial artifacts into the data, which can in turn lead to erroneous conclusions. This variability can be eliminated by sample barcoding which enables samples to be pooled, stained and run simultaneously. Existing mass cytometry barcoding approaches require time intensive labeling, reduce the number of biologically meaningful parameters and/or rely on expensive reagents. We present an approach utilizing monoisotopic cisplatin to perform cell barcoding that does not require cell permeabilization, can be completed in 10 minutes and can be utilized in combination with existing barcoding techniques to greatly increase the number of samples which can be multiplexed to improve throughput and consistency.
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Cisplatino/farmacología , Cisplatino/farmacocinética , Células Madre Embrionarias Humanas/metabolismo , Espectrometría de Masas/métodos , Tipificación Molecular/métodos , Línea Celular , Células Madre Embrionarias Humanas/citología , HumanosRESUMEN
Although overexpression/activation of focal adhesion kinase (FAK) is widely known in solid tumors to control cell growth, survival, invasion, metastasis, gene expression, and stem cell self-renewal, its expression and function in myeloid leukemia are not well investigated. Using reverse-phase protein arrays in large cohorts of newly diagnosed acute myeloid leukemia (AML) and myeloid dysplastic syndrome (MDS) samples, we found that high FAK expression was associated with unfavorable cytogenetics (P = 2 × 10-4) and relapse (P = 0.02) in AML. FAK expression was significantly lower in patients with FLT3-ITD (P = 0.0024) or RAS (P = 0.05) mutations and strongly correlated with p-SRC and integrinß3 levels. FAK protein levels were significantly higher in CD34+ (P = 5.42 × 10-20) and CD34+CD38- MDS (P = 7.62 × 10-9) cells compared with normal CD34+ cells. MDS patients with higher FAK in CD34+ cells tended to have better overall survival (P = 0.05). FAK expression was significantly higher in MDS patients who later transformed to compared with those who did not transform to AML and in AML patients who transformed from MDS compared with those with de novo AML. Coculture with mesenchymal stromal cells (MSC) increased FAK expression in AML cells. Inhibition of FAK decreased MSC-mediated adhesion/migration and viability of AML cells and prolonged survival in an AML xenograft murine model. Our results suggest that FAK regulates leukemia-stromal interactions and supports leukemia cell survival; hence, FAK is a potential therapeutic target in myeloid leukemia. Mol Cancer Ther; 16(6); 1133-44. ©2017 AACR.
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Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Leucemia Mieloide Aguda/metabolismo , Síndromes Mielodisplásicos/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Animales , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Biomarcadores , Adhesión Celular , Comunicación Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Modelos Animales de Enfermedad , Femenino , Proteína-Tirosina Quinasas de Adhesión Focal/antagonistas & inhibidores , Proteína-Tirosina Quinasas de Adhesión Focal/genética , Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/tratamiento farmacológico , Masculino , Persona de Mediana Edad , Terapia Molecular Dirigida , Síndromes Mielodisplásicos/diagnóstico , Síndromes Mielodisplásicos/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Transducción de Señal , Células del Estroma/efectos de los fármacos , Células del Estroma/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Anti-angiogenesis therapy has shown clinical benefit in patients with high-grade serous ovarian cancer (HGSC), but adaptive resistance rapidly emerges. Thus, approaches to overcome such resistance are needed. We developed the setting of adaptive resistance to anti-VEGF therapy, and performed a series of in vivo experiments in both immune competent and nude mouse models. Given the pro-angiogenic properties of tumor-associated macrophages (TAMs) and the dominant role of CSF1R in macrophage function, we added CSF1R inhibitors following emergence of adaptive resistance to anti-VEGF antibody. Mice treated with a CSF1R inhibitor (AC708) after anti-VEGF antibody resistance had little to no measurable tumor burden upon completion of the experiment while those that did not receive a CSF1R inhibitor still had abundant tumor. To mimic clinically used regimens, mice were also treated with anti-VEGF antibody and paclitaxel until resistance emerged, and then a CSF1R inhibitor was added. The addition of a CSF1R inhibitor restored response to anti-angiogenesis therapy, resulting in 83% lower tumor burden compared to treatment with anti-VEGF antibody and paclitaxel alone. Collectively, our data demonstrate that the addition of a CSF1R inhibitor to anti-VEGF therapy and taxane chemotherapy results in robust anti-tumor effects.
RESUMEN
To better understand how the apoptosis repressor with caspase recruitment domain (ARC) protein confers drug resistance in acute myeloid leukemia (AML), we investigated the role of ARC in regulating leukemia-mesenchymal stromal cell (MSC) interactions. In addition to the previously reported effect on AML apoptosis, we have demonstrated that ARC enhances migration and adhesion of leukemia cells to MSCs both in vitro and in a novel human extramedullary bone/bone marrow mouse model. Mechanistic studies revealed that ARC induces IL1ß expression in AML cells and increases CCL2, CCL4, and CXCL12 expression in MSCs, both through ARC-mediated activation of NFκB. Expression of these chemokines in MSCs increased by AML cells in an ARC/IL1ß-dependent manner; likewise, IL1ß expression was elevated when leukemia cells were co-cultured with MSCs. Further, cells from AML patients expressed the receptors for and migrated toward CCL2, CCL4, and CXCL12. Inhibition of IL1ß suppressed AML cell migration and sensitized the cells co-cultured with MSCs to chemotherapy. Our results suggest the existence of a complex ARC-regulated circuit that maintains intimate connection of AML with the tumor microenvironment through NFκB/IL1ß-regulated chemokine receptor/ligand axes and reciprocal crosstalk resulting in cytoprotection. The data implicate ARC as a promising drug target to potentially sensitize AML cells to chemotherapy.
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Proteínas del Citoesqueleto/metabolismo , Resistencia a Antineoplásicos , Interleucina-1beta/metabolismo , Leucemia Mieloide Aguda/patología , Células Madre Mesenquimatosas/patología , FN-kappa B/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Microambiente Tumoral/efectos de los fármacos , Animales , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/metabolismo , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones SCID , Transducción de Señal/efectos de los fármacos , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BCR-ABL tyrosine kinase inhibitors (TKIs) are effective against chronic myeloid leukemia (CML), but they rarely eliminate CML stem cells. Disease relapse is common upon therapy cessation, even in patients with complete molecular responses. Furthermore, once CML progresses to blast crisis (BC), treatment outcomes are dismal. We hypothesized that concomitant targeting of BCL-2 and BCR-ABL tyrosine kinase could overcome these limitations. We demonstrate increased BCL-2 expression at the protein level in bone marrow cells, particularly in Lin(-)Sca-1(+)cKit(+) cells of inducible CML in mice, as determined by CyTOF mass cytometry. Further, selective inhibition of BCL-2, aided by TKI-mediated MCL-1 and BCL-XL inhibition, markedly decreased leukemic Lin(-)Sca-1(+)cKit(+) cell numbers and long-term stem cell frequency and prolonged survival in a murine CML model. Additionally, this combination effectively eradicated CD34(+)CD38(-), CD34(+)CD38(+), and quiescent stem/progenitor CD34(+) cells from BC CML patient samples. Our results suggest that BCL-2 is a key survival factor for CML stem/progenitor cells and that combined inhibition of BCL-2 and BCR-ABL tyrosine kinase has the potential to significantly improve depth of response and cure rates of chronic-phase and BC CML.
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Proteínas de Fusión bcr-abl/antagonistas & inhibidores , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Células Madre Neoplásicas/patología , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Animales , Antígenos CD34/metabolismo , Apoptosis/efectos de los fármacos , Crisis Blástica/tratamiento farmacológico , Crisis Blástica/patología , Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Proteínas de Fusión bcr-abl/metabolismo , Humanos , Ratones Transgénicos , Células Madre Neoplásicas/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismoRESUMEN
mTOR activation leads to enhanced survival signaling in acute myeloid leukemia (AML) cells. The active-site mTOR inhibitors (asTORi) represent a promising new approach to targeting mTOR in AKT/mTOR signaling. MLN0128 is an orally-administered, second-generation asTORi, currently in clinical development. We examined the anti-leukemic effects and the mechanisms of action of MLN0128 in AML cell lines and primary samples, with a particular focus on its effect in AML stem/progenitor cells. MLN0128 inhibited cell proliferation and induced apoptosis in AML by attenuating the activity of mTOR complex 1 and 2. Using time-of-flight mass cytometry, we demonstrated that MLN0128 selectively targeted and functionally inhibited AML stem/progenitor cells with high AKT/mTOR signaling activity. Using the reverse-phase protein array technique, we measured expression and phosphorylation changes in response to MLN0128 in 151 proteins from 24 primary AML samples and identified several pro-survival pathways that antagonize MLN0128-induced cellular stress. A combined blockade of AKT/mTOR signaling and these pro-survival pathways facilitated AML cell killing. Our findings provide a rationale for the clinical use of MLN0128 to target AML and AML stem/progenitor cells, and support the use of combinatorial multi-targeted approaches in AML therapy.