RESUMEN
Tau aggregate-bearing lesions are pathological markers and potential mediators of tauopathic neurodegenerative diseases, including Alzheimer's disease. The molecular chaperone DJ-1 colocalizes with tau pathology in these disorders, but it has been unclear what functional link exists between them. In this study, we examined the consequences of tau/DJ-1 interaction as isolated proteins in vitro. When added to full-length 2N4R tau under aggregation-promoting conditions, DJ-1 inhibited both the rate and extent of filament formation in a concentration-dependent manner. Inhibitory activity was low affinity, did not require ATP, and was not affected by substituting oxidation incompetent missense mutation C106A for wild-type DJ-1. In contrast, missense mutations previously linked to familial Parkinson's disease and loss of α-synuclein chaperone activity, M26I and E64D, displayed diminished tau chaperone activity relative to wild-type DJ-1. Although DJ-1 directly bound the isolated microtubule-binding repeat region of tau protein, exposure of preformed tau seeds to DJ-1 did not diminish seeding activity in a biosensor cell model. These data reveal DJ-1 to be a holdase chaperone capable of engaging tau as a client in addition to α-synuclein. Our findings support a role for DJ-1 as part of an endogenous defense against the aggregation of these intrinsically disordered proteins.
Asunto(s)
Enfermedades Neurodegenerativas , Enfermedad de Parkinson , Humanos , alfa-Sinucleína/química , Enfermedad de Parkinson/metabolismo , Proteínas tau/genética , Chaperonas Moleculares/genética , Proteína Desglicasa DJ-1/genéticaRESUMEN
An ideal fluorescent dye for staining cell organelles should have multiple properties including specificity, stability, biocompatibility, and a large Stokes shift. Tunable photophysical properties enable 1,8-naphthalimide to serve as an excellent fluorophore in biomedical applications. Many naphthalimide derivatives have been developed into drugs, sensors, and other dyes. In this study, a series of 1,8-naphthalimide derivatives targeting live cell mitochondria were synthesized. Among these probes, Mt-4 was characterized as the best one, with highly specific mitochondrial localization, low cytotoxicity, and a large Stokes shift. More importantly, Mt-4 stood out as a potential mitochondrial dye for living-cell experiments involving induced mitochondrial stress arising from the treatments because Mt-4 shows enhanced fluorescence in mitochondrial stress situations.
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Colorantes Fluorescentes/química , Mitocondrias/metabolismo , Naftalimidas/química , Supervivencia Celular , Colorantes Fluorescentes/síntesis química , Células HeLa , Humanos , Estructura MolecularRESUMEN
Acute myeloid leukemia (AML) is a highly aggressive hematologic cancer with poor survival across a broad range of molecular subtypes. Development of efficacious and well-tolerable therapies encompassing the range of mutations that can arise in AML remains an unmet need. The bromo- and extra-terminal domain (BET) family of proteins represents an attractive therapeutic target in AML due to their crucial roles in many cellular functions, regardless of any specific mutation. Many BET inhibitors (BETi) are currently in pre-clinical and early clinical development, but acquisition of resistance continues to remain an obstacle for the drug class. Novel methods to circumvent this development of resistance could be instrumental for the future use of BET inhibitors in AML, both as monotherapy and in combination. To date, many investigations into possible drug combinations of BETi with CDK inhibitors have focused on CDK9, which has a known physical and functional interaction with the BET protein BRD4. Therefore, we wished to investigate possible synergy and additive effects between inhibitors of these targets in AML. Here, we describe combination therapy with the multi-CDK inhibitor dinaciclib and the BETi PLX51107 in pre-clinical models of AML. Dinaciclib and PLX51107 demonstrate additive effects in AML cell lines, primary AML samples, and in vivo. Further, we demonstrate novel activity of dinaciclib through inhibition of the canonical/ß-catenin dependent Wnt signaling pathway, a known resistance mechanism to BETi in AML. We show dinaciclib inhibits Wnt signaling at multiple levels, including downregulation of ß-catenin, the Wnt co-receptor LRP6, as well as many Wnt pathway components and targets. Moreover, dinaciclib sensitivity remains unaffected in a setting of BET resistance, demonstrating similar inhibitory effects on Wnt signaling when compared to BET-sensitive cells. Ultimately, our results demonstrate rationale for combination CDKi and BETi in AML. In addition, our novel finding of Wnt signaling inhibition could have potential implications in other cancers where Wnt signaling is dysregulated and demonstrates one possible approach to circumvent development of BET resistance in AML.
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Chronic lymphocytic leukemia (CLL) is effectively treated with targeted therapies including Bruton tyrosine kinase inhibitors and BCL2 antagonists. When these become ineffective, treatment options are limited. Positive transcription elongation factor complex (P-TEFb), a heterodimeric protein complex composed of cyclin dependent kinase 9 (CDK9) and cyclin T1, functions to regulate short half-life transcripts by phosphorylation of RNA Polymerase II (POLII). These transcripts are frequently dysregulated in hematologic malignancies; however, therapies targeting inhibition of P-TEFb have not yet achieved approval for cancer treatment. VIP152 kinome profiling revealed CDK9 as the main enzyme inhibited at 100 nM, with over a 10-fold increase in potency compared with other inhibitors currently in development for this target. VIP152 induced cell death in CLL cell lines and primary patient samples. Transcriptome analysis revealed inhibition of RNA degradation through the AU-Rich Element (ARE) dysregulation. Mechanistically, VIP152 inhibits the assembly of P-TEFb onto the transcription machinery and disturbs binding partners. Finally, immune competent mice engrafted with CLL-like cells of Eµ-MTCP1 over-expressing mice and treated with VIP152 demonstrated reduced disease burden and improvement in overall survival compared to vehicle-treated mice. These data suggest that VIP152 is a highly selective inhibitor of CDK9 that represents an attractive new therapy for CLL.
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Leucemia Linfocítica Crónica de Células B , Factor B de Elongación Transcripcional Positiva , Animales , Ratones , Factor B de Elongación Transcripcional Positiva/metabolismo , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Linfocítica Crónica de Células B/metabolismo , Quinasa 9 Dependiente de la Ciclina , Ciclina T/metabolismo , Fosforilación , Núcleo Celular/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéuticoRESUMEN
Altered hematopoietic stem cell (HSC) fate underlies primary blood disorders but microenvironmental factors controlling this are poorly understood. Genetically barcoded genome editing of synthetic target arrays for lineage tracing (GESTALT) zebrafish were used to screen for factors expressed by the sinusoidal vascular niche that alter the phylogenetic distribution of the HSC pool under native conditions. Dysregulated expression of protein kinase C delta (PKC-δ, encoded by prkcda) increases the number of HSC clones by up to 80% and expands polyclonal populations of immature neutrophil and erythroid precursors. PKC agonists such as cxcl8 augment HSC competition for residency within the niche and expand defined niche populations. CXCL8 induces association of PKC-δ with the focal adhesion complex, activating extracellular signal-regulated kinase (ERK) signaling and expression of niche factors in human endothelial cells. Our findings demonstrate the existence of reserve capacity within the niche that is controlled by CXCL8 and PKC and has significant impact on HSC phylogenetic and phenotypic fate.
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Células Endoteliales , Pez Cebra , Animales , Humanos , Células Endoteliales/metabolismo , Hematopoyesis/genética , Células Madre Hematopoyéticas/metabolismo , Filogenia , Proteína Quinasa C-delta/metabolismo , Nicho de Células Madre , Interleucina-8/metabolismoRESUMEN
Richter's Transformation (RT) is a poorly understood and fatal progression of chronic lymphocytic leukemia (CLL) manifesting histologically as diffuse large B-cell lymphoma. Protein arginine methyltransferase 5 (PRMT5) is implicated in lymphomagenesis, but its role in CLL or RT progression is unknown. We demonstrate herein that tumors uniformly overexpress PRMT5 in patients with progression to RT. Furthermore, mice with B-specific overexpression of hPRMT5 develop a B-lymphoid expansion with increased risk of death, and Eµ-PRMT5/TCL1 double transgenic mice develop a highly aggressive disease with transformation that histologically resembles RT; where large-scale transcriptional profiling identifies oncogenic pathways mediating PRMT5-driven disease progression. Lastly, we report the development of a SAM-competitive PRMT5 inhibitor, PRT382, with exclusive selectivity and optimal in vitro and in vivo activity compared to available PRMT5 inhibitors. Taken together, the discovery that PRMT5 drives oncogenic pathways promoting RT provides a compelling rationale for clinical investigation of PRMT5 inhibitors such as PRT382 in aggressive CLL/RT cases.
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Leucemia Linfocítica Crónica de Células B , Linfoma de Células B Grandes Difuso , Animales , Ratones , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/patología , Linfoma de Células B Grandes Difuso/patologíaRESUMEN
Using a genome-wide CRISPR screen, we identified CDK9, DHODH, and PRMT5 as synthetic lethal partners with gilteritinib treatment in fms-like tyrosine kinase 3 (FLT3)-internal tandem duplication (ITD) acute myeloid leukemia (AML) and genetically and pharmacologically validated their roles in gilteritinib sensitivity. The presence of FLT3-ITD is associated with an increase in anaerobic glycolysis, rendering leukemia cells highly sensitive to inhibition of glycolysis. Supportive of this, our data show the enrichment of single guide RNAs targeting 28 glycolysis-related genes upon gilteritinib treatment, suggesting that switching from glycolysis to oxidative phosphorylation (OXPHOS) may represent a metabolic adaption of AML in gilteritinib resistance. CDK9i/FLT3i, DHODHi/FLT3i, and PRMT5i/FLT3i pairs mechanistically converge on OXPHOS and purine biosynthesis blockade, implying that targeting the metabolic functions of these three genes and/or proteins may represent attractive strategies to sensitize AML to gilteritinib treatment. Our findings provide the basis for maximizing therapeutic impact of FLT3-ITD inhibitors and a rationale for a clinical trial of these novel combinations.
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Hematopoiesis is hierarchical, and it has been postulated that acute myeloid leukemia (AML) is organized similarly with leukemia stem cells (LSCs) residing at the apex. Limited cells acquired by fluorescence activated cell sorting in tandem with targeted amplicon-based sequencing (LC-FACSeq) enables identification of mutations in small subpopulations of cells, such as LSCs. Leveraging this, we studied clonal compositions of immunophenotypically-defined compartments in AML through genomic and functional analyses at diagnosis, remission and relapse in 88 AML patients. Mutations involving DNA methylation pathways, transcription factors and spliceosomal machinery did not differ across compartments, while signaling pathway mutations were less frequent in putative LSCs. We also provide insights into TP53-mutated AML by demonstrating stepwise acquisition of mutations beginning from the preleukemic hematopoietic stem cell stage. In 10 analyzed cases, acquisition of additional mutations and del(17p) led to genetic and functional heterogeneity within the LSC pool with subclones harboring varying degrees of clonogenic potential. Finally, we use LC-FACSeq to track clonal evolution in serial samples, which can also be a powerful tool to direct targeted therapy against measurable residual disease. Therefore, studying clinically significant small subpopulations of cells can improve our understanding of AML biology and offers advantages over bulk sequencing to monitor the evolution of disease.
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Biomarcadores de Tumor/genética , Evolución Clonal , Genómica/métodos , Células Madre Hematopoyéticas/patología , Leucemia Mieloide Aguda/patología , Mutación , Células Madre Neoplásicas/patología , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Citometría de Flujo , Estudios de Seguimiento , Células Madre Hematopoyéticas/metabolismo , Humanos , Inmunofenotipificación , Leucemia Mieloide Aguda/genética , Masculino , Persona de Mediana Edad , Células Madre Neoplásicas/metabolismo , Pronóstico , Adulto JovenRESUMEN
Rare, recurrent balanced translocations occur in a variety of cancers but are often not functionally interrogated. Balanced translocations with the immunoglobulin heavy chain locus (IGH; 14q32) in chronic lymphocytic leukemia (CLL) are infrequent but have led to the discovery of pathogenic genes including CCND1, BCL2, and BCL3. Following identification of a t(X;14)(q28;q32) translocation that placed the mature T cell proliferation 1 gene (MTCP1) adjacent to the immunoglobulin locus in a CLL patient, we hypothesized that this gene may have previously unrecognized importance. Indeed, here we report overexpression of human MTCP1 restricted to the B cell compartment in mice produces a clonal CD5+/CD19+ leukemia recapitulating the major characteristics of human CLL and demonstrates favorable response to therapeutic intervention with ibrutinib. We reinforce the importance of genetic interrogation of rare, recurrent balanced translocations to identify cancer driving genes via the story of MTCP1 as a contributor to CLL pathogenesis.
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Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Translocación Genética , Adulto , Anciano , Anciano de 80 o más Años , Animales , Proteínas del Linfoma 3 de Células B , Ciclina D1 , Femenino , Regulación de la Expresión Génica , Genes de las Cadenas Pesadas de las Inmunoglobulinas , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Oncogenes/genética , Proteínas Proto-Oncogénicas c-bcl-2RESUMEN
PURPOSE: Nicotinamide phosphoribosyltransferase (NAMPT) inhibitors (NAMPTi) are currently in development, but may be limited as single-agent therapy due to compound-specific toxicity and cancer metabolic plasticity allowing resistance development. To potentially lower the doses of NAMPTis required for therapeutic benefit against acute myeloid leukemia (AML), we performed a genome-wide CRISPRi screen to identify rational disease-specific partners for a novel NAMPTi, KPT-9274. EXPERIMENTAL DESIGN: Cell lines and primary cells were analyzed for cell viability, self-renewal, and responses at RNA and protein levels with loss-of-function approaches and pharmacologic treatments. In vivo efficacy of combination therapy was evaluated with a xenograft model. RESULTS: We identified two histone deacetylases (HDAC), HDAC8 and SIRT6, whose knockout conferred synthetic lethality with KPT-9274 in AML. Furthermore, HDAC8-specific inhibitor, PCI-34051, or clinical class I HDAC inhibitor, AR-42, in combination with KPT-9274, synergistically decreased the survival of AML cells in a dose-dependent manner. AR-42/KPT-9274 cotreatment attenuated colony-forming potentials of patient cells while sparing healthy hematopoietic cells. Importantly, combined therapy demonstrated promising in vivo efficacy compared with KPT-9274 or AR-42 monotherapy. Mechanistically, genetic inhibition of SIRT6 potentiated the effect of KPT-9274 on PARP-1 suppression by abolishing mono-ADP ribosylation. AR-42/KPT-9274 cotreatment resulted in synergistic attenuation of homologous recombination and nonhomologous end joining pathways in cell lines and leukemia-initiating cells. CONCLUSIONS: Our findings provide evidence that HDAC8 inhibition- or shSIRT6-induced DNA repair deficiencies are potently synergistic with NAMPT targeting, with minimal toxicity toward normal cells, providing a rationale for a novel-novel combination-based treatment for AML.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Citocinas/antagonistas & inhibidores , Leucemia Mieloide Aguda/tratamiento farmacológico , Nicotinamida Fosforribosiltransferasa/antagonistas & inhibidores , Proteínas Represoras/antagonistas & inhibidores , Sirtuinas/antagonistas & inhibidores , Acrilamidas/farmacología , Acrilamidas/uso terapéutico , Aminopiridinas/farmacología , Aminopiridinas/uso terapéutico , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Línea Celular Tumoral , Daño del ADN , Reparación del ADN por Unión de Extremidades/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Técnicas de Inactivación de Genes , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Humanos , Ácidos Hidroxámicos/farmacología , Ácidos Hidroxámicos/uso terapéutico , Indoles/farmacología , Indoles/uso terapéutico , Leucemia Mieloide Aguda/patología , Masculino , Ratones , Fenilbutiratos/farmacología , Fenilbutiratos/uso terapéutico , Reparación del ADN por Recombinación/efectos de los fármacos , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Sirtuinas/genética , Sirtuinas/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Acute myeloid leukemia (AML) is a hematopoietic stem-cell-derived leukemia with often successive derived driver mutations. Late onset acquisition of internal tandem duplication in FLT3 (FLT3-ITD) at a high variant allele frequency often contributes to full transformation to a highly proliferative, rapidly progressive disease with poor outcome. The FLT3-ITD mutation is targetable with approved FLT3 small molecule inhibitors, including midostaurin and gilteritinib. However, outside of patients receiving allogeneic transplant, most patients fail to respond or relapse, suggesting alternative approaches of therapy will be required. We employed genome-wide pooled CRISPR knockout screening as a method for large-scale identification of targets whose knockout produces a phenotypic effect that enhances the antitumor properties of FLT3 inhibitors. Among the candidate targets we identified the effect of XPO1 knockout to be synergistic with midostaurin treatment. Next, we validated the genetic finding with pharmacologic combination of the slowly reversible XPO1 inhibitor selinexor with midostaurin and gilteritinib in FLT3-ITD AML cell lines and primary patient samples. Lastly, we demonstrated improved survival with either combination therapy compared to its monotherapy components in an aggressive AML murine model, supporting further evaluation and rapid clinical translation of this combination strategy.
RESUMEN
Acute myeloid leukemia (AML) is a heterogeneous and complex disease, and treatments for this disease have not been curative for the majority of patients. In younger patients, internal tandem duplication of FLT3 (FLT3-ITD) is a common mutation for which two inhibitors (midostaurin and gilteritinib) with varied potency and specificity for FLT3 are clinically approved. However, the high rate of relapse or failed initial response of AML patients suggests that the addition of a second targeted therapy may be necessary to improve efficacy. Using an unbiased large-scale CRISPR screen, we genetically identified BCL2 knockout as having synergistic effects with an approved FLT3 inhibitor. Here, we provide supportive studies that validate the therapeutic potential of the combination of FLT3 inhibitors with venetoclax in vitro and in vivo against multiple models of FLT3-ITD-driven AML. Our unbiased approach provides genetic validation for co-targeting FLT3 and BCL2 and repurposes CRISPR screening data, utilizing the genome-wide scope toward mechanistic understanding.