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1.
Blood ; 133(1): 70-80, 2019 01 03.
Artículo en Inglés | MEDLINE | ID: mdl-30322870

RESUMEN

Inhibition of the B-cell receptor (BCR) signaling pathway is a promising treatment strategy in multiple B-cell malignancies. However, the role of BCR blockade in diffuse large B-cell lymphoma (DLBCL) remains undefined. We recently characterized primary DLBCL subsets with distinct genetic bases for perturbed BCR/phosphoinositide 3-kinase (PI3K) signaling and dysregulated B-cell lymphoma 2 (BCL-2) expression. Herein, we explore the activity of PI3K inhibitors and BCL-2 blockade in a panel of functionally and genetically characterized DLBCL cell line models. A PI3K inhibitor with predominant α/δ activity, copanlisib, exhibited the highest cytotoxicity in all BCR-dependent DLBCLs. The proapoptotic effect of copanlisib was associated with DLBCL subtype-specific dysregulated expression of BCL-2 family members including harakiri (HRK) and its antiapoptotic partner BCL extra large (BCL-xL), BCL2 related protein A1, myeloid cell leukemia 1 (MCL-1), and BCL2 interacting mediator of cell death. Using functional BH3 profiling, we found that the cytotoxic activity of copanlisib was primarily mediated through BCL-xL and MCL-1-dependent mechanisms that might complement BCL-2 blockade. For these reasons, we evaluated single-agent activity of venetoclax in the DLBCLs and identified a subset with limited sensitivity to BCL-2 blockade despite having genetic bases of BCL-2 dysregulation. As these were largely BCR-dependent DLBCLs, we hypothesized that combined inhibition of PI3Kα/δ and BCL-2 would perturb BCR-dependent and BCL-2-mediated survival pathways. Indeed, we observed synergistic activity of copanlisib/venetoclax in BCR-dependent DLBCLs with genetic bases for BCL-2 dysregulation in vitro and confirmed these findings in a xenograft model. These results provide preclinical evidence for the rational combination of PI3Kα/δ and BCL-2 blockade in genetically defined DLBCLs.


Asunto(s)
Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Fosfatidilinositol 3-Quinasa Clase I/antagonistas & inhibidores , Sinergismo Farmacológico , Linfoma de Células B Grandes Difuso/patología , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Pirimidinas/farmacología , Quinazolinas/farmacología , Sulfonamidas/farmacología , Animales , Antineoplásicos/farmacología , Apoptosis , Proliferación Celular , Femenino , Humanos , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Linfoma de Células B Grandes Difuso/genética , Ratones , Ratones Endogámicos NOD , Ratones SCID , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto
2.
Proc Natl Acad Sci U S A ; 115(5): E886-E895, 2018 01 30.
Artículo en Inglés | MEDLINE | ID: mdl-29339518

RESUMEN

Bcl-2 family proteins regulate apoptosis, and aberrant interactions of overexpressed antiapoptotic family members such as Mcl-1 promote cell transformation, cancer survival, and resistance to chemotherapy. Discovering potent and selective Mcl-1 inhibitors that can relieve apoptotic blockades is thus a high priority for cancer research. An attractive strategy for disabling Mcl-1 involves using designer peptides to competitively engage its binding groove, mimicking the structural mechanism of action of native sensitizer BH3-only proteins. We transformed Mcl-1-binding peptides into α-helical, cell-penetrating constructs that are selectively cytotoxic to Mcl-1-dependent cancer cells. Critical to the design of effective inhibitors was our introduction of an all-hydrocarbon cross-link or "staple" that stabilizes α-helical structure, increases target binding affinity, and independently confers binding specificity for Mcl-1 over related Bcl-2 family paralogs. Two crystal structures of complexes at 1.4 Å and 1.9 Å resolution demonstrate how the hydrophobic staple induces an unanticipated structural rearrangement in Mcl-1 upon binding. Systematic sampling of staple location and iterative optimization of peptide sequence in accordance with established design principles provided peptides that target intracellular Mcl-1. This work provides proof of concept for the development of potent, selective, and cell-permeable stapled peptides for therapeutic targeting of Mcl-1 in cancer, applying a design and validation workflow applicable to a host of challenging biomedical targets.


Asunto(s)
Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Neoplasias/metabolismo , Péptidos/química , Animales , Sitios de Unión , Línea Celular , Supervivencia Celular , Dicroismo Circular , Cristalografía por Rayos X , Citoplasma/metabolismo , Diseño de Fármacos , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Concentración 50 Inhibidora , Ratones , Mutación , Unión Proteica , Mapeo de Interacción de Proteínas , Espectrometría de Fluorescencia
3.
Blood Cancer Discov ; 5(3): 180-201, 2024 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-38442309

RESUMEN

In many cancers, mortality is associated with the emergence of relapse with multidrug resistance (MDR). Thus far, the investigation of cancer relapse mechanisms has largely focused on acquired genetic mutations. Using acute myeloid leukemia (AML) patient-derived xenografts (PDX), we systematically elucidated a basis of MDR and identified drug sensitivity in relapsed AML. We derived pharmacologic sensitivity for 22 AML PDX models using dynamic BH3 profiling (DBP), together with genomics and transcriptomics. Using in vivo acquired resistant PDXs, we found that resistance to unrelated, narrowly targeted agents in distinct PDXs was accompanied by broad resistance to drugs with disparate mechanisms. Moreover, baseline mitochondrial apoptotic priming was consistently reduced regardless of the class of drug-inducing selection. By applying DBP, we identified drugs showing effective in vivo activity in resistant models. This study implies evasion of apoptosis drives drug resistance and demonstrates the feasibility of the DBP approach to identify active drugs for patients with relapsed AML. SIGNIFICANCE: Acquired resistance to targeted therapy remains challenging in AML. We found that reduction in mitochondrial priming and common transcriptomic signatures was a conserved mechanism of acquired resistance across different drug classes in vivo. Drugs active in vivo can be identified even in the multidrug resistant state by DBP.


Asunto(s)
Apoptosis , Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos , Leucemia Mieloide Aguda , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/patología , Leucemia Mieloide Aguda/genética , Humanos , Apoptosis/efectos de los fármacos , Animales , Ratones , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Resistencia a Múltiples Medicamentos/genética , Resistencia a Múltiples Medicamentos/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto , Células Precursoras de Granulocitos/efectos de los fármacos , Células Precursoras de Granulocitos/patología , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico
4.
Proc Natl Acad Sci U S A ; 107(29): 12895-900, 2010 Jul 20.
Artículo en Inglés | MEDLINE | ID: mdl-20615979

RESUMEN

It is well established that CD4(+) CD8(+) thymocytes are more sensitive to myriad death stimuli than CD4(+) or CD8(+) single positive (SP) thymocytes. The mechanism behind this hypersensitivity to apoptosis of CD4(+) CD8(+) thymocytes is not understood. To test whether the difference lay in the apoptotic preset of mitochondria, established by the BCL-2 family of proteins, we developed a method, FACS-based BH3 profiling. Using this tool, we could discriminate thymocyte subpopulations and demonstrate that mitochondria in double positive (DP) thymocytes are more primed for death than those in single positive counterparts. Loss of proapoptotic BIM, known to cause autoimmunity, also causes loss of "priming." Priming is a phenotype with physiologic consequences, which can be measured at the single-cell level in complex samples using FACS-based BH3 profiling.


Asunto(s)
Apoptosis/inmunología , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Mitocondrias/inmunología , Timo/citología , Animales , Línea Celular , Permeabilidad de la Membrana Celular , Reactividad Cruzada/inmunología , Citometría de Flujo , Humanos , Potencial de la Membrana Mitocondrial , Ratones , Membranas Mitocondriales/metabolismo , Fragmentos de Péptidos/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Timo/inmunología
5.
Cancer Discov ; 12(2): 432-449, 2022 02.
Artículo en Inglés | MEDLINE | ID: mdl-34531254

RESUMEN

CRISPR-Cas9-based genetic screens have successfully identified cell type-dependent liabilities in cancer, including acute myeloid leukemia (AML), a devastating hematologic malignancy with poor overall survival. Because most of these screens have been performed in vitro using established cell lines, evaluating the physiologic relevance of these targets is critical. We have established a CRISPR screening approach using orthotopic xenograft models to validate and prioritize AML-enriched dependencies in vivo, including in CRISPR-competent AML patient-derived xenograft (PDX) models tractable for genome editing. Our integrated pipeline has revealed several targets with translational value, including SLC5A3 as a metabolic vulnerability for AML addicted to exogenous myo-inositol and MARCH5 as a critical guardian to prevent apoptosis in AML. MARCH5 repression enhanced the efficacy of BCL2 inhibitors such as venetoclax, further highlighting the clinical potential of targeting MARCH5 in AML. Our study provides a valuable strategy for discovery and prioritization of new candidate AML therapeutic targets. SIGNIFICANCE: There is an unmet need to improve the clinical outcome of AML. We developed an integrated in vivo screening approach to prioritize and validate AML dependencies with high translational potential. We identified SLC5A3 as a metabolic vulnerability and MARCH5 as a critical apoptosis regulator in AML, both of which represent novel therapeutic opportunities.This article is highlighted in the In This Issue feature, p. 275.


Asunto(s)
Antineoplásicos/uso terapéutico , Sistemas CRISPR-Cas , Leucemia Mieloide Aguda/tratamiento farmacológico , Medicina de Precisión , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Humanos , Leucemia Mieloide Aguda/genética
6.
Signal Transduct Target Ther ; 7(1): 51, 2022 02 21.
Artículo en Inglés | MEDLINE | ID: mdl-35185150

RESUMEN

Despite high initial response rates, acute myeloid leukemia (AML) treated with the BCL-2-selective inhibitor venetoclax (VEN) alone or in combinations commonly acquires resistance. We performed gene/protein expression, metabolomic and methylation analyses of isogenic AML cell lines sensitive or resistant to VEN, and identified the activation of RAS/MAPK pathway, leading to increased stability and higher levels of MCL-1 protein, as a major acquired mechanism of VEN resistance. MCL-1 sustained survival and maintained mitochondrial respiration in VEN-RE cells, which had impaired electron transport chain (ETC) complex II activity, and MCL-1 silencing or pharmacologic inhibition restored VEN sensitivity. In support of the importance of RAS/MAPK activation, we found by single-cell DNA sequencing rapid clonal selection of RAS-mutated clones in AML patients treated with VEN-containing regimens. In summary, these findings establish RAS/MAPK/MCL-1 and mitochondrial fitness as key survival mechanisms of VEN-RE AML and provide the rationale for combinatorial strategies effectively targeting these pathways.


Asunto(s)
Compuestos Bicíclicos Heterocíclicos con Puentes , Leucemia Mieloide Aguda , Sistema de Señalización de MAP Quinasas , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Proteínas Proto-Oncogénicas c-bcl-2 , Sulfonamidas , Proteínas ras , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Línea Celular Tumoral , Resistencia a Antineoplásicos , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Sulfonamidas/farmacología
7.
Blood Cancer Discov ; 2(1): 70-91, 2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-33447829

RESUMEN

Based on gene expression profiles, diffuse large B cell lymphoma (DLBCL) is sub-divided into germinal center B cell-like (GCB) and activated B cell-like (ABC) DLBCL. Two of the most common genomic aberrations in ABC-DLBCL are mutations in MYD88, as well as BCL2 copy number gains. Here, we employ immune phenotyping, RNA-Seq and whole exome sequencing to characterize a Myd88 and Bcl2-driven mouse model of ABC-DLBCL. We show that this model resembles features of human ABC-DLBCL. We further demonstrate an actionable dependence of our murine ABC-DLBCL model on BCL2. This BCL2 dependence was also detectable in human ABC-DLBCL cell lines. Moreover, human ABC-DLBCLs displayed increased PD-L1 expression, compared to GCB-DLBCL. In vivo experiments in our ABC-DLBCL model showed that combined venetoclax and RMP1-14 significantly increased the overall survival of lymphoma bearing animals, indicating that this combination may be a viable option for selected human ABC-DLBCL cases harboring MYD88 and BCL2 aberrations.


Asunto(s)
Linfoma de Células B Grandes Difuso , Factor 88 de Diferenciación Mieloide , Animales , Genes bcl-2 , Centro Germinal/metabolismo , Linfoma de Células B Grandes Difuso/genética , Ratones , Factor 88 de Diferenciación Mieloide/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética
8.
Cancer Cell ; 38(6): 872-890.e6, 2020 12 14.
Artículo en Inglés | MEDLINE | ID: mdl-33217342

RESUMEN

Acquired resistance to BH3 mimetic antagonists of BCL-2 and MCL-1 is an important clinical problem. Using acute myelogenous leukemia (AML) patient-derived xenograft (PDX) models of acquired resistance to BCL-2 (venetoclax) and MCL-1 (S63845) antagonists, we identify common principles of resistance and persistent vulnerabilities to overcome resistance. BH3 mimetic resistance is characterized by decreased mitochondrial apoptotic priming as measured by BH3 profiling, both in PDX models and human clinical samples, due to alterations in BCL-2 family proteins that vary among cases, but not to acquired mutations in leukemia genes. BCL-2 inhibition drives sequestered pro-apoptotic proteins to MCL-1 and vice versa, explaining why in vivo combinations of BCL-2 and MCL-1 antagonists are more effective when concurrent rather than sequential. Finally, drug-induced mitochondrial priming measured by dynamic BH3 profiling (DBP) identifies drugs that are persistently active in BH3 mimetic-resistant myeloblasts, including FLT-3 inhibitors and SMAC mimetics.


Asunto(s)
Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Resistencia a Antineoplásicos , Leucemia Mieloide Aguda/patología , Mitocondrias/metabolismo , Pirimidinas/farmacología , Sulfonamidas/farmacología , Tiofenos/farmacología , Animales , Apoptosis , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/metabolismo , Ratones , Mitocondrias/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Proteínas Proto-Oncogénicas/farmacología , Transducción de Señal
9.
Elife ; 62017 06 08.
Artículo en Inglés | MEDLINE | ID: mdl-28594323

RESUMEN

Overexpression of anti-apoptotic Bcl-2 family proteins contributes to cancer progression and confers resistance to chemotherapy. Small molecules that target Bcl-2 are used in the clinic to treat leukemia, but tight and selective inhibitors are not available for Bcl-2 paralog Bfl-1. Guided by computational analysis, we designed variants of the native BH3 motif PUMA that are > 150-fold selective for Bfl-1 binding. The designed peptides potently trigger disruption of the mitochondrial outer membrane in cells dependent on Bfl-1, but not in cells dependent on other anti-apoptotic homologs. High-resolution crystal structures show that designed peptide FS2 binds Bfl-1 in a shifted geometry, relative to PUMA and other binding partners, due to a set of epistatic mutations. FS2 modified with an electrophile reacts with a cysteine near the peptide-binding groove to augment specificity. Designed Bfl-1 binders provide reagents for cellular profiling and leads for developing enhanced and cell-permeable peptide or small-molecule inhibitors.


Asunto(s)
Proteínas Reguladoras de la Apoptosis/genética , Mutación , Fragmentos de Péptidos/genética , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/genética , Proteínas Reguladoras de la Apoptosis/química , Proteínas Reguladoras de la Apoptosis/metabolismo , Cristalografía por Rayos X , Antígenos de Histocompatibilidad Menor , Modelos Moleculares , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Unión Proteica , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/metabolismo
10.
Cancer Cell ; 31(1): 142-156, 2017 01 09.
Artículo en Inglés | MEDLINE | ID: mdl-28017613

RESUMEN

It is not understood why healthy tissues can exhibit varying levels of sensitivity to the same toxic stimuli. Using BH3 profiling, we find that mitochondria of many adult somatic tissues, including brain, heart, and kidneys, are profoundly refractory to pro-apoptotic signaling, leading to cellular resistance to cytotoxic chemotherapies and ionizing radiation. In contrast, mitochondria from these tissues in young mice and humans are primed for apoptosis, predisposing them to undergo cell death in response to genotoxic damage. While expression of the apoptotic protein machinery is nearly absent by adulthood, in young tissues its expression is driven by c-Myc, linking developmental growth to cell death. These differences may explain why pediatric cancer patients have a higher risk of developing treatment-associated toxicities.


Asunto(s)
Apoptosis , Mitocondrias/fisiología , Neoplasias/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-myc/fisiología , Factores de Edad , Animales , Doxorrubicina/toxicidad , Humanos , Ratones , Neoplasias/patología , Especificidad de Órganos , Proteína Destructora del Antagonista Homólogo bcl-2/fisiología , Proteína X Asociada a bcl-2/fisiología
12.
ACS Chem Biol ; 11(5): 1238-44, 2016 05 20.
Artículo en Inglés | MEDLINE | ID: mdl-26854535

RESUMEN

Alpha helices form a critical part of the binding interface for many protein-protein interactions, and chemically stabilized synthetic helical peptides can be effective inhibitors of such helix-mediated complexes. In particular, hydrocarbon stapling of peptides to generate constrained helices can improve binding affinity and other peptide properties, but determining the best stapled peptide variant often requires laborious trial and error. Here, we describe the rapid discovery and optimization of a stapled-helix peptide that binds to Mcl-1, an antiapoptotic protein that is overexpressed in many chemoresistant cancers. To accelerate discovery, we developed a peptide library synthesis and screening scheme capable of identifying subtle affinity differences among Mcl-1-binding stapled peptides. We used our method to sample combinations of non-natural amino-acid substitutions that we introduced into Mcl-1 inhibitors in the context of a fixed helix-stabilizing hydrocarbon staple that increased peptide helical content and reduced proteolysis. Peptides discovered in our screen contained surprising substitutions at sites that are conserved in natural binding partners. Library-identified peptide M3d is the most potent molecule yet tested for selectively triggering mitochondrial permeabilization in Mcl-1 dependent cell lines. Our library approach for optimizing helical peptide inhibitors can be readily applied to the study of other biomedically important targets.


Asunto(s)
Proteína 1 de la Secuencia de Leucemia de Células Mieloides/antagonistas & inhibidores , Péptidos/química , Péptidos/farmacología , Secuencia de Aminoácidos , Animales , Línea Celular Tumoral , Descubrimiento de Drogas , Humanos , Ratones , Membranas Mitocondriales/efectos de los fármacos , Simulación del Acoplamiento Molecular , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Biblioteca de Péptidos , Estructura Secundaria de Proteína
13.
J Clin Invest ; 126(10): 3827-3836, 2016 10 03.
Artículo en Inglés | MEDLINE | ID: mdl-27599292

RESUMEN

Upfront resistance to chemotherapy and relapse following remission are critical problems in leukemia that are generally attributed to subpopulations of chemoresistant tumor cells. There are, however, limited means for prospectively identifying these subpopulations, which hinders an understanding of therapeutic resistance. BH3 profiling is a functional single-cell analysis using synthetic BCL-2 BH3 domain-like peptides that measures mitochondrial apoptotic sensitivity or "priming." Here, we observed that the extent of apoptotic priming is heterogeneous within multiple cancer cell lines and is not the result of experimental noise. Apoptotic priming was also heterogeneous in treatment-naive primary human acute myeloid leukemia (AML) myeloblasts, and this heterogeneity decreased in chemotherapy-treated AML patients. The priming of the most apoptosis-resistant tumor cells, rather than the median priming of the population, best predicted patient response to induction chemotherapy. For several patients, these poorly primed subpopulations of AML tumor cells were enriched for antiapoptotic proteins. Developing techniques to identify and understand these apoptosis-insensitive subpopulations of tumor cells may yield insights into clinical chemoresistance and potentially improve therapeutic outcomes in AML.


Asunto(s)
Apoptosis/efectos de los fármacos , Leucemia Mieloide Aguda/patología , Antineoplásicos Fitogénicos/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Resistencia a Antineoplásicos , Ensayos de Selección de Medicamentos Antitumorales , Etopósido/farmacología , Células Precursoras de Granulocitos/efectos de los fármacos , Células Precursoras de Granulocitos/fisiología , Humanos , Concentración 50 Inhibidora , Leucemia Mieloide Aguda/tratamiento farmacológico , Mitocondrias/efectos de los fármacos , Análisis de la Célula Individual
14.
Cancer Cell ; 28(1): 29-41, 2015 Jul 13.
Artículo en Inglés | MEDLINE | ID: mdl-26175414

RESUMEN

A variety of cancers depend on JAK2 signaling, including the high-risk subset of B cell acute lymphoblastic leukemias (B-ALLs) with CRLF2 rearrangements. Type I JAK2 inhibitors induce paradoxical JAK2 hyperphosphorylation in these leukemias and have limited activity. To improve the efficacy of JAK2 inhibition in B-ALL, we developed the type II inhibitor CHZ868, which stabilizes JAK2 in an inactive conformation. CHZ868 potently suppressed the growth of CRLF2-rearranged human B-ALL cells, abrogated JAK2 signaling, and improved survival in mice with human or murine B-ALL. CHZ868 and dexamethasone synergistically induced apoptosis in JAK2-dependent B-ALLs and further improved in vivo survival compared to CHZ868 alone. These data support the testing of type II JAK2 inhibition in patients with JAK2-dependent leukemias and other disorders.


Asunto(s)
Aminopiridinas/administración & dosificación , Antineoplásicos/administración & dosificación , Bencimidazoles/administración & dosificación , Dexametasona/administración & dosificación , Resistencia a Antineoplásicos/efectos de los fármacos , Janus Quinasa 2/antagonistas & inhibidores , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/administración & dosificación , Aminopiridinas/farmacología , Animales , Antineoplásicos/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Apoptosis , Bencimidazoles/farmacología , Línea Celular Tumoral , Citoprotección/efectos de los fármacos , Sinergismo Farmacológico , Humanos , Janus Quinasa 2/química , Janus Quinasa 2/genética , Ratones , Mutación , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Inhibidores de Proteínas Quinasas/farmacología , Transducción de Señal/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto
15.
ACS Chem Biol ; 9(9): 1962-8, 2014 Sep 19.
Artículo en Inglés | MEDLINE | ID: mdl-25052212

RESUMEN

Mcl-1 is overexpressed in many cancers and can confer resistance to cell-death signaling in refractory disease. Molecules that specifically inhibit Mcl-1 hold potential for diagnosing and disrupting Mcl-1-dependent cell survival. We selected three peptides from a yeast-surface display library that showed moderate specificity and affinity for binding to Mcl-1 over Bfl-1, Bcl-xL, Bcl-2, and Bcl-w. Specificity for Mcl-1 was improved by introducing threonine at peptide position 2e. The most specific peptide, MS1, bound Mcl-1 with 40-fold or greater specificity over four other human Bcl-2 paralogs. In BH3 profiling assays, MS1 caused depolarization in several human Mcl-1-dependent cell lines with EC50 values of ∼3 µM, contrasted with EC50 values of >100 µM for Bcl-2-, Bcl-xL-, or Bfl-1-dependent cell lines. MS1 is at least 30-fold more potent in this assay than the previously used Mcl-1 targeting reagent NoxaA BH3. These peptides can be used to detect Mcl-1 dependency in cells and provide leads for developing Mcl-1 targeting therapeutics.


Asunto(s)
Terapia Molecular Dirigida/métodos , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Fragmentos de Péptidos/metabolismo , Péptidos/química , Péptidos/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Reguladoras de la Apoptosis/química , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína 11 Similar a Bcl2 , Línea Celular/efectos de los fármacos , Técnicas de Visualización de Superficie Celular/métodos , Polarización de Fluorescencia , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Antígenos de Histocompatibilidad Menor , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/antagonistas & inhibidores , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Péptidos/farmacología , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Treonina/química , Treonina/metabolismo , Proteína bcl-X/metabolismo
16.
Cell Stem Cell ; 13(4): 483-91, 2013 Oct 03.
Artículo en Inglés | MEDLINE | ID: mdl-23954752

RESUMEN

Human embryonic stem cells (hESCs) are highly sensitive to DNA damage and have low survival ability relative to differentiated cells. We investigated the source of this difference by comparing damage response pathways in hESCs and differentiated cells. We found that hESCs undergo more rapid p53-dependent apoptosis after DNA damage than differentiated cells do. However, p53 localization and function are similar between hESCs and differentiated cells, suggesting that p53 alone cannot explain the difference in sensitivity. Instead, we show that mitochondrial readiness for apoptosis, known as mitochondrial priming, differs between hESCs and differentiated cells. Specifically, the balance between proapoptotic and antiapoptotic proteins is shifted closer to the apoptotic threshold in hESCs than in differentiated cells. Altering this balance in differentiated cells increases their sensitivity and results in cell death, suggesting that manipulation of mitochondrial priming could potentially alter the sensitivity of other stem cells, including cancer stem cells.


Asunto(s)
Apoptosis , Daño del ADN , Células Madre Embrionarias/citología , Mitocondrias/metabolismo , Células Madre Embrionarias/metabolismo , Humanos , Proteína p53 Supresora de Tumor/metabolismo
17.
Science ; 334(6059): 1129-33, 2011 Nov 25.
Artículo en Inglés | MEDLINE | ID: mdl-22033517

RESUMEN

Cytotoxic chemotherapy targets elements common to all nucleated human cells, such as DNA and microtubules, yet it selectively kills tumor cells. Here we show that clinical response to these drugs correlates with, and may be partially governed by, the pretreatment proximity of tumor cell mitochondria to the apoptotic threshold, a property called mitochondrial priming. We used BH3 profiling to measure priming in tumor cells from patients with multiple myeloma, acute myelogenous and lymphoblastic leukemia, and ovarian cancer. This assay measures mitochondrial response to peptides derived from proapoptotic BH3 domains of proteins critical for death signaling to mitochondria. Patients with highly primed cancers exhibited superior clinical response to chemotherapy. In contrast, chemoresistant cancers and normal tissues were poorly primed. Manipulation of mitochondrial priming might enhance the efficacy of cytotoxic agents.


Asunto(s)
Antineoplásicos/uso terapéutico , Apoptosis , Mitocondrias/fisiología , Neoplasias/tratamiento farmacológico , Neoplasias/fisiopatología , Adulto , Anciano , Animales , Línea Celular Tumoral , Proliferación Celular , Niño , Supervivencia sin Enfermedad , Resistencia a Antineoplásicos , Femenino , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/fisiopatología , Masculino , Potencial de la Membrana Mitocondrial , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/fisiopatología , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/fisiopatología , Fragmentos de Péptidos/metabolismo , Permeabilidad , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/fisiopatología , Proteínas Proto-Oncogénicas c-bcl-2/química , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Inducción de Remisión , Transducción de Señal
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