RESUMEN
Heat shock protein 90 (HSP90) is a chaperone with vital roles in regulating proteostasis, long recognized for its function in protein folding and maturation. A view is emerging that identifies HSP90 not as one protein that is structurally and functionally homogeneous but, rather, as a protein that is shaped by its environment. In this Review, we discuss evidence of multiple structural forms of HSP90 in health and disease, including homo-oligomers and hetero-oligomers, also termed epichaperomes, and examine the impact of stress, post-translational modifications and co-chaperones on their formation. We describe how these variations influence context-dependent functions of HSP90 as well as its interaction with other chaperones, co-chaperones and proteins, and how this structural complexity of HSP90 impacts and is impacted by its interaction with small molecule modulators. We close by discussing recent developments regarding the use of HSP90 inhibitors in cancer and how our new appreciation of the structural and functional heterogeneity of HSP90 invites a re-evaluation of how we discover and implement HSP90 therapeutics for disease treatment.
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Proteínas HSP90 de Choque Térmico , Chaperonas Moleculares , Proteínas HSP90 de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , Pliegue de Proteína , Proteostasis , HomeostasisRESUMEN
The tolerance of amino acid starvation is fundamental to robust cellular fitness. Asparagine depletion is lethal to some cancer cells, a vulnerability that can be exploited clinically. We report that resistance to asparagine starvation is uniquely dependent on an N-terminal low-complexity domain of GSK3α, which its paralog GSK3ß lacks. In response to depletion of specific amino acids, including asparagine, leucine, and valine, this domain mediates supramolecular assembly of GSK3α with ubiquitin-proteasome system components in spatially sequestered cytoplasmic bodies. This effect is independent of mTORC1 or GCN2. In normal cells, GSK3α promotes survival during essential amino acid starvation. In human leukemia, GSK3α body formation predicts asparaginase resistance, and sensitivity to asparaginase combined with a GSK3α inhibitor. We propose that GSK3α body formation provides a cellular mechanism to maximize the catalytic efficiency of proteasomal protein degradation in response to amino acid starvation, an adaptive response co-opted by cancer cells for asparaginase resistance.
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Asparaginasa , Leucemia , Aminoácidos/metabolismo , Asparaginasa/genética , Asparaginasa/metabolismo , Asparaginasa/farmacología , Asparagina , Humanos , Proteínas Serina-Treonina QuinasasRESUMEN
Post-translational modifications such as protein N-glycosylation, significantly influence cellular processes. Dysregulated N-glycosylation, exemplified in Grp94, a member of the Hsp90 family, leads to structural changes and the formation of epichaperomes, contributing to pathologies. Targeting N-glycosylation-induced conformations offers opportunities for developing selective chemical tools and drugs for these pathologic forms of chaperones. We here demonstrate how a specific Grp94 conformation induced by N-glycosylation, identified previously via molecular dynamics simulations, rationalizes the distinct behavior of similar ligands. Integrating dynamic ligand unbinding information with SAR development, we differentiate ligands productively engaging the pathologic Grp94 conformers from those that are not. Additionally, analyzing binding site stereoelectronic properties and QSAR models using cytotoxicity data unveils relationships between chemical, conformational properties, and biological activities. These findings facilitate the design of ligands targeting specific Grp94 conformations induced by abnormal glycosylation, selectively disrupting pathogenic protein networks while sparing normal mechanisms.
RESUMEN
Under normal conditions, heat shock proteins work in unison through dynamic protein interactions collectively referred to as the "chaperome." Recent work revealed that during cellular stress, the functional interactions of the chaperome are modified to form the "epichaperome," which results in improper protein folding, degradation, aggregation, and transport. This study is the first to investigate this novel mechanism of protein dishomeostasis in traumatic brain injury (TBI). Male and female adult, Sprague-Dawley rats received a lateral controlled cortical impact (CCI) and the ipsilateral hippocampus was collected 24 h 1, 2, and 4 weeks after injury. The epichaperome complex was visualized by measuring HSP90, HSC70 and HOP expression in native-PAGE and normalized to monomeric protein expression. A two-way ANOVA examined the effect of injury and sex at each time-point. Native HSP90, HSC70 and HOP protein expression showed a significant effect of injury effect across all time-points. Additionally, HSC70 and HOP showed significant sex effects at 24 h and 4 weeks. Altogether, controlled cortical impact significantly increased formation of the epichaperome across all proteins measured. Further investigation of this pathological mechanism can lead to a greater understanding of the link between TBI and increased risk of neurodegenerative disease and targeting the epichaperome for therapeutics.
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Lesiones Traumáticas del Encéfalo , Enfermedades Neurodegenerativas , Femenino , Masculino , Ratas , Animales , Ratas Sprague-Dawley , Análisis de Varianza , HipocampoRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is typically diagnosed at an advanced stage, which limits surgical options and portends a dismal prognosis. Current oncologic PDAC therapies confer marginal benefit and, thus, a significant unmet clinical need exists for new therapeutic strategies. To identify effective PDAC therapies, we leveraged a syngeneic orthotopic PDAC transplant mouse model to perform a large-scale, in vivo screen of 16 single-agent and 41 two-drug targeted therapy combinations in mice. Among 57 drug conditions screened, combined inhibition of heat shock protein (Hsp)-90 and MEK was found to produce robust suppression of tumor growth, leading to an 80% increase in the survival of PDAC-bearing mice with no significant toxicity. Mechanistically, we observed that single-agent MEK inhibition led to compensatory activation of resistance pathways, including components of the PI3K/AKT/mTOR signaling axis, which was overcome with the addition of HSP90 inhibition. The combination of HSP90(i) + MEK(i) was also active in vitro in established human PDAC cell lines and in vivo in patient-derived organoid PDAC transplant models. These findings encourage the clinical development of HSP90(i) + MEK(i) combination therapy and highlight the power of clinically relevant in vivo model systems for identifying cancer therapies.
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Antineoplásicos/farmacología , Evaluación Preclínica de Medicamentos , Ensayos de Selección de Medicamentos Antitumorales , Adenocarcinoma/metabolismo , Adenocarcinoma/mortalidad , Adenocarcinoma/patología , Animales , Antineoplásicos/uso terapéutico , Benzodioxoles/farmacología , Biomarcadores de Tumor , Línea Celular Tumoral , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos/métodos , Ensayos de Selección de Medicamentos Antitumorales/métodos , Sinergismo Farmacológico , Expresión Génica , Humanos , Inmunohistoquímica , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Terapia Molecular Dirigida , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/mortalidad , Neoplasias Pancreáticas/patología , Inhibidores de Proteínas Quinasas/farmacología , Purinas/farmacología , Piridonas/farmacología , Pirimidinonas/farmacología , Transducción de Señal/efectos de los fármacos , Tasa de Supervivencia , Resultado del Tratamiento , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Transient, multi-protein complexes are important facilitators of cellular functions. This includes the chaperome, an abundant protein family comprising chaperones, co-chaperones, adaptors, and folding enzymes-dynamic complexes of which regulate cellular homeostasis together with the protein degradation machinery. Numerous studies have addressed the role of chaperome members in isolation, yet little is known about their relationships regarding how they interact and function together in malignancy. As function is probably highly dependent on endogenous conditions found in native tumours, chaperomes have resisted investigation, mainly due to the limitations of methods needed to disrupt or engineer the cellular environment to facilitate analysis. Such limitations have led to a bottleneck in our understanding of chaperome-related disease biology and in the development of chaperome-targeted cancer treatment. Here we examined the chaperome complexes in a large set of tumour specimens. The methods used maintained the endogenous native state of tumours and we exploited this to investigate the molecular characteristics and composition of the chaperome in cancer, the molecular factors that drive chaperome networks to crosstalk in tumours, the distinguishing factors of the chaperome in tumours sensitive to pharmacologic inhibition, and the characteristics of tumours that may benefit from chaperome therapy. We find that under conditions of stress, such as malignant transformation fuelled by MYC, the chaperome becomes biochemically 'rewired' to form a network of stable, survival-facilitating, high-molecular-weight complexes. The chaperones heat shock protein 90 (HSP90) and heat shock cognate protein 70 (HSC70) are nucleating sites for these physically and functionally integrated complexes. The results indicate that these tightly integrated chaperome units, here termed the epichaperome, can function as a network to enhance cellular survival, irrespective of tissue of origin or genetic background. The epichaperome, present in over half of all cancers tested, has implications for diagnostics and also provides potential vulnerability as a target for drug intervention.
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Chaperonas Moleculares/metabolismo , Complejos Multiproteicos/metabolismo , Neoplasias/metabolismo , Neoplasias/patología , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Descubrimiento de Drogas , Femenino , Genes myc/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Ratones , Chaperonas Moleculares/antagonistas & inhibidores , Complejos Multiproteicos/antagonistas & inhibidores , Complejos Multiproteicos/química , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Especificidad de ÓrganosRESUMEN
Adaptation to acute and chronic stress and/or persistent stressors is a subject of wide interest in central nervous system disorders. In this context, stress is an effector of change in organismal homeostasis and the response is generated when the brain perceives a potential threat. Herein, we discuss a nuanced and granular view whereby a wide variety of genotoxic and environmental stressors, including aging, genetic risk factors, environmental exposures, and age- and lifestyle-related changes, act as direct insults to cellular, as opposed to organismal, homeostasis. These two concepts of how stressors impact the central nervous system are not mutually exclusive. We discuss how maladaptive stressor-induced changes in protein connectivity through epichaperomes, disease-associated pathologic scaffolds composed of tightly bound chaperones, co-chaperones, and other factors, impact intracellular protein functionality altering phenotypes, that in turn disrupt and remodel brain networks ranging from intercellular to brain connectome levels. We provide an evidence-based view on how these maladaptive changes ranging from stressor to phenotype provide unique precision medicine opportunities for diagnostic and therapeutic development, especially in the context of neurodegenerative disorders including Alzheimer's disease where treatment options are currently limited.
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Envejecimiento/metabolismo , Encéfalo/metabolismo , Exposición a Riesgos Ambientales/efectos adversos , Chaperonas Moleculares/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Plasticidad Neuronal/fisiología , Adaptación Fisiológica/fisiología , Envejecimiento/patología , Animales , Encéfalo/patología , Chaperonina 60/metabolismo , Respuesta al Choque Térmico/fisiología , Homeostasis/fisiología , Humanos , Enfermedades Neurodegenerativas/patología , Estrés Oxidativo/fisiologíaRESUMEN
The repurposing of existing drugs has emerged as an attractive additional strategy to the development of novel compounds in the fight against cancerous diseases. Inhibition of phosphodiesterase 5 (PDE5) has been claimed as a potential approach to target various cancer subtypes in recent years. However, data on the treatment of tumors with PDE5 inhibitors as well as the underlying mechanisms are as yet very scarce. Here, we report that treatment of tumor cells with low concentrations of Sildenafil was associated with decreased cancer cell proliferation and augmented apoptosis in vitro and resulted in impaired tumor growth in vivo. Notably, incubation of cancer cells with Sildenafil was associated with altered expression of HSP90 chaperone followed by degradation of protein kinase D2, a client protein previously reported to be involved in tumor growth. Furthermore, the involvement of low doses of PU-H71, an HSP90 inhibitor currently under clinical evaluation, in combination with low concentrations of Sildenafil, synergistically and negatively impacted on the viability of cancer cells in vivo. Taken together, our study suggests that repurposing of already approved drugs, alone or in combination with oncology-dedicated compounds, may represent a novel cancer therapeutic strategy.
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Proteínas HSP90 de Choque Térmico/metabolismo , Neoplasias/patología , Inhibidores de Fosfodiesterasa 5/farmacología , Proteolisis , Citrato de Sildenafil/farmacología , Canales Catiónicos TRPP/metabolismo , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Humanos , Neoplasias/metabolismoRESUMEN
The hsp90 chaperones govern the function of essential client proteins critical for normal cell function as well as cancer initiation and progression. Hsp90 activity is driven by ATP, which binds to the N-terminal domain and induces large conformational changes that are required for client maturation. Inhibitors targeting the ATP-binding pocket of the N-terminal domain have anticancer effects, but most bind with similar affinity to cytosolic Hsp90α and Hsp90ß, endoplasmic reticulum Grp94, and mitochondrial Trap1, the four cellular hsp90 paralogs. Paralog-specific inhibitors may lead to drugs with fewer side effects. The ATP-binding pockets of the four paralogs are flanked by three side pockets, termed sites 1, 2, and 3, which differ between the paralogs in their accessibility to inhibitors. Previous insights into the principles governing access to sites 1 and 2 have resulted in development of paralog-selective inhibitors targeting these sites, but the rules for selective targeting of site 3 are less clear. Earlier studies identified 5'N-ethylcarboxamido adenosine (NECA) as a Grp94-selective ligand. Here we use NECA and its derivatives to probe the properties of site 3. We found that derivatives that lengthen the 5' moiety of NECA improve selectivity for Grp94 over Hsp90α. Crystal structures reveal that the derivatives extend further into site 3 of Grp94 compared with their parent compound and that selectivity is due to paralog-specific differences in ligand pose and ligand-induced conformational strain in the protein. These studies provide a structural basis for Grp94-selective inhibition using site 3.
Asunto(s)
Adenosina-5'-(N-etilcarboxamida)/farmacología , Glicoproteínas de Membrana/química , Simulación del Acoplamiento Molecular , Adenosina-5'-(N-etilcarboxamida)/análogos & derivados , Regulación Alostérica , Sitios de Unión , Humanos , Glicoproteínas de Membrana/metabolismo , Unión ProteicaRESUMEN
The chaperome is the collection of proteins in the cell that carry out molecular chaperoning functions. Changes in the interaction strength between chaperome proteins lead to an assembly that is functionally and structurally distinct from each constituent member. In this review, we discuss the epichaperome, the cellular network that forms when the chaperome components of distinct chaperome machineries come together as stable, functionally integrated, multimeric complexes. In tumors, maintenance of the epichaperome network is vital for tumor survival, rendering them vulnerable to therapeutic interventions that target critical epichaperome network components. We discuss how the epichaperome empowers an approach for precision medicine cancer trials where a new target, biomarker, and relevant drug candidates can be correlated and integrated. We introduce chemical biology methods to investigate the heterogeneity of the chaperome in a given cellular context. Lastly, we discuss how ligand-protein binding kinetics are more appropriate than equilibrium binding parameters to characterize and unravel chaperome targeting in cancer and to gauge the selectivity of ligands for specific tumor-associated chaperome pools.
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Antineoplásicos , Sistemas de Liberación de Medicamentos/métodos , Chaperonas Moleculares , Proteínas de Neoplasias , Neoplasias , Mapas de Interacción de Proteínas/efectos de los fármacos , Animales , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapéutico , Humanos , Chaperonas Moleculares/antagonistas & inhibidores , Chaperonas Moleculares/metabolismo , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Neoplasias/patologíaRESUMEN
The chaperome is a large family of proteins composed of chaperones, co-chaperones and a multitude of other factors. Elegant studies in yeast and other organisms have paved the road to how we currently understand the complex organization of this large family into protein networks. The goal of this chapter is to provide an overview of chaperome networks in cancer cells, with a focus on two cellular states defined by chaperome network organization. One state characterized by chaperome networks working in isolation and with little overlap, contains global chaperome networks resembling those of normal, non-transformed, cells. We propose that in this state, redundancy in chaperome networks results in a tumor type unamenable for single-agent chaperome therapy. The second state comprises chaperome networks interconnected in response to cellular stress, such as MYC hyperactivation. This is a state where no redundant pathways can be deployed, and is a state of vulnerability, amenable for chaperome therapy. We conclude by proposing a change in how we discover and implement chaperome inhibitor strategies, and suggest an approach to chaperome therapy where the properties of chaperome networks, rather than genetics or client proteins, are used in chaperome inhibitor implementation.
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Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Chaperonas Moleculares/antagonistas & inhibidores , Chaperonas Moleculares/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Humanos , Neoplasias/patologíaRESUMEN
Hsp90α and Hsp90ß are implicated in a number of cancers and neurodegenerative disorders but the lack of selective pharmacological probes confounds efforts to identify their individual roles. Here, we analyzed the binding of an Hsp90α-selective PU compound, PU-11-trans, to the two cytosolic paralogs. We determined the co-crystal structures of Hsp90α and Hsp90ß bound to PU-11-trans, as well as the structure of the apo Hsp90ß NTD. The two inhibitor-bound structures reveal that Ser52, a nonconserved residue in the ATP binding pocket in Hsp90α, provides additional stability to PU-11-trans through a water-mediated hydrogen-bonding network. Mutation of Ser52 to alanine, as found in Hsp90ß, alters the dissociation constant of Hsp90α for PU-11-trans to match that of Hsp90ß. Our results provide a structural explanation for the binding preference of PU inhibitors for Hsp90α and demonstrate that the single nonconserved residue in the ATP-binding pocket may be exploited for α/ß selectivity.
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Aminoácidos/metabolismo , Descubrimiento de Drogas , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Proteínas HSP90 de Choque Térmico/metabolismo , Purinas/metabolismo , Secuencia de Aminoácidos , Aminoácidos/química , Aminoácidos/genética , Desarrollo de Medicamentos , Proteínas HSP90 de Choque Térmico/química , Proteínas HSP90 de Choque Térmico/genética , Humanos , Mutación , Conformación Proteica , Purinas/química , Homología de SecuenciaRESUMEN
Aggressive double- and triple-hit (DH/TH) diffuse large B-cell lymphomas (DLBCLs) feature activation of Hsp90 stress pathways. Herein, we show that Hsp90 controls posttranscriptional dynamics of key messenger RNA (mRNA) species including those encoding BCL6, MYC, and BCL2. Using a proteomics approach, we found that Hsp90 binds to and maintains activity of eIF4E. eIF4E drives nuclear export and translation of BCL6, MYC, and BCL2 mRNA. eIF4E RNA-immunoprecipitation sequencing in DLBCL suggests that nuclear eIF4E controls an extended program that includes B-cell receptor signaling, cellular metabolism, and epigenetic regulation. Accordingly, eIF4E was required for survival of DLBCL including the most aggressive subtypes, DH/TH lymphomas. Indeed, eIF4E inhibition induces tumor regression in cell line and patient-derived tumorgrafts of TH-DLBCL, even in the presence of elevated Hsp90 activity. Targeting Hsp90 is typically limited by counterregulatory elevation of Hsp70B, which induces resistance to Hsp90 inhibitors. Surprisingly, we identify Hsp70 mRNA as an eIF4E target. In this way, eIF4E inhibition can overcome drug resistance to Hsp90 inhibitors. Accordingly, rational combinatorial inhibition of eIF4E and Hsp90 inhibitors resulted in cooperative antilymphoma activity in DH/TH DLBCL in vitro and in vivo.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Núcleo Celular/metabolismo , Linfoma de Células B/tratamiento farmacológico , Linfoma de Células B/metabolismo , Proteínas de Neoplasias/antagonistas & inhibidores , ARN Mensajero/metabolismo , ARN Neoplásico/metabolismo , Transporte Activo de Núcleo Celular/efectos de los fármacos , Línea Celular Tumoral , Núcleo Celular/patología , Humanos , Linfoma de Células B/patología , Proteínas de Neoplasias/metabolismoRESUMEN
The identification of somatic activating mutations in JAK2 (refs 14) and in the thrombopoietin receptor gene (MPL) in most patients with myeloproliferative neoplasm (MPN) led to the clinical development of JAK2 kinase inhibitors. JAK2 inhibitor therapy improves MPN-associated splenomegaly and systemic symptoms but does not significantly decrease or eliminate the MPN clone in most patients with MPN. We therefore sought to characterize mechanisms by which MPN cells persist despite chronic inhibition of JAK2. Here we show that JAK2 inhibitor persistence is associated with reactivation of JAKSTAT signalling and with heterodimerization between activated JAK2 and JAK1 or TYK2, consistent with activation of JAK2 in trans by other JAK kinases. Further, this phenomenon is reversible: JAK2 inhibitor withdrawal is associated with resensitization to JAK2 kinase inhibitors and with reversible changes in JAK2 expression. We saw increased JAK2 heterodimerization and sustained JAK2 activation in cell lines, in murine models and in patients treated with JAK2 inhibitors. RNA interference and pharmacological studies show that JAK2-inhibitor-persistent cells remain dependent on JAK2 protein expression. Consequently, therapies that result in JAK2 degradation retain efficacy in persistent cells and may provide additional benefit to patients with JAK2-dependent malignancies treated with JAK2 inhibitors.
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Janus Quinasa 2/antagonistas & inhibidores , Janus Quinasa 2/metabolismo , Trastornos Mieloproliferativos/tratamiento farmacológico , Multimerización de Proteína , Factores de Transcripción STAT/metabolismo , Transducción de Señal , Animales , Línea Celular , Modelos Animales de Enfermedad , Resistencia a Antineoplásicos/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Granulocitos/efectos de los fármacos , Granulocitos/enzimología , Granulocitos/metabolismo , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Janus Quinasa 1/biosíntesis , Janus Quinasa 1/deficiencia , Janus Quinasa 1/genética , Janus Quinasa 1/metabolismo , Janus Quinasa 2/deficiencia , Janus Quinasa 2/genética , Ratones , Trastornos Mieloproliferativos/enzimología , Trastornos Mieloproliferativos/metabolismo , Trastornos Mieloproliferativos/patología , Fosforilación , Biosíntesis de Proteínas , Interferencia de ARN , Transducción de Señal/efectos de los fármacos , TYK2 Quinasa/biosíntesis , TYK2 Quinasa/deficiencia , TYK2 Quinasa/genética , TYK2 Quinasa/metabolismoRESUMEN
INTRODUCTION: Heat shock protein 90 (HSP90) regulates protein homeostasis in eukaryotes. As a 'professional interactor', HSP90 binds to and chaperones many proteins and has both housekeeping and disease-related functions but its regulation remains in part elusive. HSP90 complexes are a target for therapy, notably against cancer, and several inhibitors are currently in clinical trials. Proteomic studies have revealed the vast interaction network of HSP90 and, in doing so, the extent of cellular processes the chaperone takes part in, especially in yeast and human cells. Furthermore, small-molecule inhibitors were used to probe the global impact of its inhibition on the proteome. Areas covered: We review here recent HSP90-related interactomics and total proteome studies and their relevance for research on cancer, neurodegenerative and pathogen diseases. Expert commentary: Proteomics experiments are our best chance to identify the context-dependent global proteome of HSP90 and thus uncover and understand its disease-specific biology. However, understanding the complexity of HSP90 will require multiple complementary, quantitative approaches and novel bioinformatics to translate interactions into ordered functional networks and pathways. Developing therapies will necessitate more knowledge on HSP90 complexes and networks with disease relevance and on total proteome changes induced by their perturbation. Most work has been done in cancer, thus a lot remains to be done in the context of other diseases.
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Proteínas HSP90 de Choque Térmico/metabolismo , Interacciones Huésped-Patógeno , Neoplasias/tratamiento farmacológico , Enfermedades Neurodegenerativas/tratamiento farmacológico , Proteómica/métodos , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Humanos , Neoplasias/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Procesamiento Proteico-PostraduccionalRESUMEN
The development of the dual Janus kinase 1/2 (JAK1/2) inhibitor ruxolitinib for the treatment of myeloproliferative neoplasms (MPNs) has led to studies of ruxolitinib in other clinical contexts, including JAK-mutated acute lymphoblastic leukemia (ALL). However, the limited ability of JAK inhibition to induce molecular or clinicopathological responses in MPNs suggests a need for development of better therapies for JAK kinase-dependent malignancies. Here, we demonstrate that heat shock protein 90 (HSP90) inhibition using a purine-scaffold HSP90 inhibitor in early clinical development is an effective therapeutic approach in JAK-dependent ALL and can overcome persistence to JAK-inhibitor therapy in ALL cells.
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Benzodioxoles/farmacología , Proteínas HSP90 de Choque Térmico , Janus Quinasa 1 , Janus Quinasa 2 , Proteínas de Neoplasias , Leucemia-Linfoma Linfoblástico de Células Precursoras , Purinas/farmacología , Animales , Femenino , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Proteínas HSP90 de Choque Térmico/genética , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Janus Quinasa 1/genética , Janus Quinasa 1/metabolismo , Janus Quinasa 2/genética , Janus Quinasa 2/metabolismo , Masculino , Ratones , Mutación , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
A copper-mediated synthesis of diaryl sulfides utilizing Cu(I)-thiophene-2-carboxylate (CuTC) is described. We demonstrate the use of CuTC as a soluble, non-basic catalyst in the coupling of aryl iodides and aryl thiols in the synthesis of synthetically advanced diaryl sulfides. This method allows for the successful coupling of challenging substrates including ortho-substituted and heteroaryl iodides and thiols. Additionally, most of the aryl iodide substrates used here contain the privileged piperazine scaffold bound to a pyrimidine, pyridine, or phenyl ring and thus this method allows for the elaboration of complex piperazine scaffolds into molecules of biological interest. The method described here enables the incorporation of late-stage structural diversity into diaryl sulfides containing the piperazine ring, thus enhancing the number and nature of derivatives available for SAR investigation.
RESUMEN
Patients with myeloproliferative neoplasms (MPNs) are at significant, cumulative risk of leukemic transformation to acute myeloid leukemia (AML), which is associated with adverse clinical outcome and resistance to standard AML therapies. We performed genomic profiling of post-MPN AML samples; these studies demonstrate somatic tumor protein 53 (TP53) mutations are common in JAK2V617F-mutant, post-MPN AML but not in chronic-phase MPN and lead to clonal dominance of JAK2V617F/TP53-mutant leukemic cells. Consistent with these data, expression of JAK2V617F combined with Tp53 loss led to fully penetrant AML in vivo. JAK2V617F-mutant, Tp53-deficient AML was characterized by an expanded megakaryocyte erythroid progenitor population that was able to propagate the disease in secondary recipients. In vitro studies revealed that post-MPN AML cells were sensitive to decitabine, the JAK1/2 inhibitor ruxolitinib, or the heat shock protein 90 inhibitor 8-(6-iodobenzo[d][1.3]dioxol-5-ylthio)-9-(3-(isopropylamino)propyl)-9H-purine-6-amine (PU-H71). Treatment with ruxolitinib or PU-H71 improved survival of mice engrafted with JAK2V617F-mutant, Tp53-deficient AML, demonstrating therapeutic efficacy for these targeted therapies and providing a rationale for testing these therapies in post-MPN AML.
Asunto(s)
Neoplasias Hematológicas/complicaciones , Janus Quinasa 2/genética , Leucemia Mieloide Aguda/genética , Trastornos Mieloproliferativos/complicaciones , Trastornos Mieloproliferativos/genética , Proteína p53 Supresora de Tumor/genética , Animales , Azacitidina/análogos & derivados , Azacitidina/farmacología , Benzodioxoles/farmacología , Western Blotting , Ensayo de Unidades Formadoras de Colonias , Decitabina , Exoma/genética , Citometría de Flujo , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/etiología , Ratones , Mutación Missense/genética , Nitrilos , Purinas/farmacología , Pirazoles/farmacología , PirimidinasRESUMEN
The emerging field of RNA nanotechnology has been used to design well-programmed, self-assembled nanostructures for applications in chemistry, biology, and medicine. At the forefront of its utility in cancer is the unrestricted ability to self-assemble multiple siRNAs within a single nanostructure formulation for the RNAi screening of a wide range of oncogenes while potentiating the gene therapy of malignant tumors. In our RNAi nanotechnology approach, V- and Y-shape RNA templates were designed and constructed for the self-assembly of discrete, higher-ordered siRNA nanostructures targeting the oncogenic glucose regulated chaperones. The GRP78-targeting siRNAs self-assembled into genetically encoded spheres, triangles, squares, pentagons and hexagons of discrete sizes and shapes according to TEM imaging. Furthermore, gel electrophoresis, thermal denaturation, and CD spectroscopy validated the prerequisite siRNA hybrids for their RNAi application. In a 24 sample siRNA screen conducted within the AN3CA endometrial cancer cells known to overexpress oncogenic GRP78 activity, the self-assembled siRNAs targeting multiple sites of GRP78 expression demonstrated more potent and long-lasting anticancer activity relative to their linear controls. Extending the scope of our RNAi screening approach, the self-assembled siRNA hybrids (5 nM) targeting of GRP-75, 78, and 94 resulted in significant (50-95%) knockdown of the glucose regulated chaperones, which led to synergistic effects in tumor cell cycle arrest (50-80%) and death (50-60%) within endometrial (AN3CA), cervical (HeLa), and breast (MDA-MB-231) cancer cell lines. Interestingly, a nontumorigenic lung (MRC5) cell line displaying normal glucose regulated chaperone levels was found to tolerate siRNA treatment and demonstrated less toxicity (5-20%) relative to the cancer cells that were found to be addicted to glucose regulated chaperones. These remarkable self-assembled siRNA nanostructures may thus encompass a new class of potent siRNAs that may be useful in screening important oncogene targets while improving siRNA therapeutic efficacy and specificity in cancer.
RESUMEN
The discovery of JAK2/MPL mutations in patients with myeloproliferative neoplasms (MPN) led to clinical development of Janus kinase (JAK) inhibitors for treatment of MPN. These inhibitors improve constitutional symptoms and splenomegaly but do not significantly reduce mutant allele burden in patients. We recently showed that chronic exposure to JAK inhibitors results in inhibitor persistence via JAK2 transactivation and persistent JAK-signal transducer and activator of transcription signaling. We performed genetic and pharmacologic studies to determine whether improved JAK2 inhibition would show increased efficacy in MPN models and primary samples. Jak2 deletion in vivo led to profound reduction in disease burden not seen with JAK inhibitors, and deletion of Jak2 following chronic ruxolitinib therapy markedly reduced mutant allele burden. This demonstrates that JAK2 remains an essential target in MPN cells that survive in the setting of chronic JAK inhibition. Combination therapy with the heat shock protein 90 (HSP90) inhibitor PU-H71 and ruxolitinib reduced total and phospho-JAK2 and achieved more potent inhibition of downstream signaling than ruxolitinib monotherapy. Combination treatment improved blood counts, spleen weights, and reduced bone marrow fibrosis compared with ruxolitinib alone. These data suggest alternate approaches that increase JAK2 targeting, including combination JAK/HSP90 inhibitor therapy, are warranted in the clinical setting.