RESUMEN
DNA mismatch repair deficiency (MMRd) is associated with a high tumor mutational burden (TMB) and sensitivity to immune checkpoint blockade (ICB) therapy. Nevertheless, most MMRd tumors do not durably respond to ICB and critical questions remain about immunosurveillance and TMB in these tumors. In the present study, we developed autochthonous mouse models of MMRd lung and colon cancer. Surprisingly, these models did not display increased T cell infiltration or ICB response, which we showed to be the result of substantial intratumor heterogeneity of mutations. Furthermore, we found that immunosurveillance shapes the clonal architecture but not the overall burden of neoantigens, and T cell responses against subclonal neoantigens are blunted. Finally, we showed that clonal, but not subclonal, neoantigen burden predicts ICB response in clinical trials of MMRd gastric and colorectal cancer. These results provide important context for understanding immune evasion in cancers with a high TMB and have major implications for therapies aimed at increasing TMB.
Asunto(s)
Neoplasias Encefálicas , Neoplasias Colorrectales , Síndromes Neoplásicos Hereditarios , Animales , Ratones , Neoplasias Colorrectales/genética , Antígenos de Neoplasias/genética , Mutación , Reparación de la Incompatibilidad de ADN/genética , Biomarcadores de Tumor/genéticaRESUMEN
Lymphocytes are key for immune surveillance of tumors, but our understanding of the spatial organization and physical interactions that facilitate lymphocyte anti-cancer functions is limited. We used multiplexed imaging, quantitative spatial analysis, and machine learning to create high-definition maps of lung tumors from a Kras/Trp53-mutant mouse model and human resections. Networks of interacting lymphocytes ("lymphonets") emerged as a distinctive feature of the anti-cancer immune response. Lymphonets nucleated from small T cell clusters and incorporated B cells with increasing size. CXCR3-mediated trafficking modulated lymphonet size and number, but T cell antigen expression directed intratumoral localization. Lymphonets preferentially harbored TCF1+ PD-1+ progenitor CD8+ T cells involved in responses to immune checkpoint blockade (ICB) therapy. Upon treatment of mice with ICB or an antigen-targeted vaccine, lymphonets retained progenitor and gained cytotoxic CD8+ T cell populations, likely via progenitor differentiation. These data show that lymphonets create a spatial environment supportive of CD8+ T cell anti-tumor responses.
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Adenocarcinoma del Pulmón , Neoplasias Pulmonares , Humanos , Animales , Ratones , Linfocitos T CD8-positivos , Inmunoterapia/métodos , Neoplasias Pulmonares/patología , Adenocarcinoma del Pulmón/genética , InmunidadRESUMEN
Diverse processes in cancer are mediated by enzymes, which most proximally exert their function through their activity. High-fidelity methods to profile enzyme activity are therefore critical to understanding and targeting the pathological roles of enzymes in cancer. Here, we present an integrated set of methods for measuring specific protease activities across scales, and deploy these methods to study treatment response in an autochthonous model of Alk-mutant lung cancer. We leverage multiplexed nanosensors and machine learning to analyze in vivo protease activity dynamics in lung cancer, identifying significant dysregulation that includes enhanced cleavage of a peptide, S1, which rapidly returns to healthy levels with targeted therapy. Through direct on-tissue localization of protease activity, we pinpoint S1 cleavage to the tumor vasculature. To link protease activity to cellular function, we design a high-throughput method to isolate and characterize proteolytically active cells, uncovering a pro-angiogenic phenotype in S1-cleaving cells. These methods provide a framework for functional, multiscale characterization of protease dysregulation in cancer.
Asunto(s)
Neoplasias Pulmonares , Péptido Hidrolasas , Endopeptidasas , Humanos , Neoplasias Pulmonares/genética , Péptido Hidrolasas/metabolismo , Proteolisis , Proteínas Tirosina Quinasas ReceptorasRESUMEN
Immunosurveillance of cancer requires the presentation of peptide antigens on major histocompatibility complex class I (MHC-I) molecules1-5. Current approaches to profiling of MHC-I-associated peptides, collectively known as the immunopeptidome, are limited to in vitro investigation or bulk tumour lysates, which limits our understanding of cancer-specific patterns of antigen presentation in vivo6. To overcome these limitations, we engineered an inducible affinity tag into the mouse MHC-I gene (H2-K1) and targeted this allele to the KrasLSL-G12D/+Trp53fl/fl mouse model (KP/KbStrep)7. This approach enabled us to precisely isolate MHC-I peptides from autochthonous pancreatic ductal adenocarcinoma and from lung adenocarcinoma (LUAD) in vivo. In addition, we profiled the LUAD immunopeptidome from the alveolar type 2 cell of origin up to late-stage disease. Differential peptide presentation in LUAD was not predictable by mRNA expression or translation efficiency and is probably driven by post-translational mechanisms. Vaccination with peptides presented by LUAD in vivo induced CD8+ T cell responses in naive mice and tumour-bearing mice. Many peptides specific to LUAD, including immunogenic peptides, exhibited minimal expression of the cognate mRNA, which prompts the reconsideration of antigen prediction pipelines that triage peptides according to transcript abundance8. Beyond cancer, the KbStrep allele is compatible with other Cre-driver lines to explore antigen presentation in vivo in the pursuit of understanding basic immunology, infectious disease and autoimmunity.
Asunto(s)
Antígenos de Neoplasias , Péptidos , Proteómica , Células Epiteliales Alveolares/inmunología , Animales , Presentación de Antígeno , Antígenos de Neoplasias/análisis , Antígenos de Neoplasias/química , Antígenos de Neoplasias/inmunología , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Carcinoma Ductal Pancreático/química , Carcinoma Ductal Pancreático/inmunología , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/inmunología , Neoplasias Pulmonares/química , Neoplasias Pulmonares/inmunología , Ratones , Neoplasias Pancreáticas/química , Neoplasias Pancreáticas/inmunología , Péptidos/análisis , Péptidos/química , Péptidos/inmunología , ARN MensajeroRESUMEN
Prognostically relevant RNA expression states exist in pancreatic ductal adenocarcinoma (PDAC), but our understanding of their drivers, stability, and relationship to therapeutic response is limited. To examine these attributes systematically, we profiled metastatic biopsies and matched organoid models at single-cell resolution. In vivo, we identify a new intermediate PDAC transcriptional cell state and uncover distinct site- and state-specific tumor microenvironments (TMEs). Benchmarking models against this reference map, we reveal strong culture-specific biases in cancer cell transcriptional state representation driven by altered TME signals. We restore expression state heterogeneity by adding back in vivo-relevant factors and show plasticity in culture models. Further, we prove that non-genetic modulation of cell state can strongly influence drug responses, uncovering state-specific vulnerabilities. This work provides a broadly applicable framework for aligning cell states across in vivo and ex vivo settings, identifying drivers of transcriptional plasticity and manipulating cell state to target associated vulnerabilities.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Neoplasias Pancreáticas/metabolismo , Microambiente Tumoral , Adulto , Anciano , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Análisis de la Célula IndividualRESUMEN
Immune evasion is a hallmark of cancer, and therapies that restore immune surveillance have proven highly effective in cancers with high tumor mutation burden (TMB) (e.g., those with microsatellite instability (MSI)). Whether low TMB cancers, which are largely refractory to immunotherapy, harbor potentially immunogenic neoantigens remains unclear. Here, we show that tumors from all patients with microsatellite stable (MSS) colorectal cancer (CRC) express clonal predicted neoantigens despite low TMB. Unexpectedly, these neoantigens are broadly expressed at lower levels compared to those in MSI CRC. Using a versatile platform for modulating neoantigen expression in CRC organoids and transplantation into the distal colon of mice, we show that low expression precludes productive cross priming and drives immediate T cell dysfunction. Strikingly, experimental or therapeutic rescue of priming rendered T cells capable of controlling tumors with low neoantigen expression. These findings underscore a critical role of neoantigen expression level in immune evasion and therapy response.
Asunto(s)
Neoplasias Colorrectales , Linfocitos T , Animales , Antígenos de Neoplasias/genética , Neoplasias Colorrectales/genética , Humanos , Inmunoterapia , Ratones , Inestabilidad de MicrosatélitesRESUMEN
CD8 T cell responses against different tumor neoantigens occur simultaneously, yet little is known about the interplay between responses and its impact on T cell function and tumor control. In mouse lung adenocarcinoma, we found that immunodominance is established in tumors, wherein CD8 T cell expansion is predominantly driven by the antigen that most stably binds MHC. T cells responding to subdominant antigens were enriched for a TCF1+ progenitor phenotype correlated with response to immune checkpoint blockade (ICB) therapy. However, the subdominant T cell response did not preferentially benefit from ICB due to a dysfunctional subset of TCF1+ cells marked by CCR6 and Tc17 differentiation. Analysis of human samples and sequencing datasets revealed that CCR6+ TCF1+ cells exist across human cancers and are not correlated with ICB response. Vaccination eliminated CCR6+ TCF1+ cells and dramatically improved the subdominant response, highlighting a strategy to optimally engage concurrent neoantigen responses against tumors.
Asunto(s)
Adenocarcinoma del Pulmón/inmunología , Antígenos de Neoplasias/inmunología , Linfocitos T CD8-positivos/inmunología , Factor Nuclear 1-alfa del Hepatocito/metabolismo , Neoplasias Pulmonares/inmunología , Células Madre/inmunología , Secuencia de Aminoácidos , Animales , Antígeno CTLA-4/metabolismo , Epítopos , Femenino , Humanos , Inhibidores de Puntos de Control Inmunológico/farmacología , Neoplasias Pulmonares/patología , Ratones , Péptidos/química , Fenotipo , Receptor de Muerte Celular Programada 1/metabolismo , RNA-Seq , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores CCR6/metabolismo , Análisis de la Célula Individual , VacunaciónRESUMEN
The CD155/TIGIT axis can be co-opted during immune evasion in chronic viral infections and cancer. Pancreatic adenocarcinoma (PDAC) is a highly lethal malignancy, and immune-based strategies to combat this disease have been largely unsuccessful to date. We corroborate prior reports that a substantial portion of PDAC harbors predicted high-affinity MHC class I-restricted neoepitopes and extend these findings to advanced/metastatic disease. Using multiple preclinical models of neoantigen-expressing PDAC, we demonstrate that intratumoral neoantigen-specific CD8+ T cells adopt multiple states of dysfunction, resembling those in tumor-infiltrating lymphocytes of PDAC patients. Mechanistically, genetic and/or pharmacologic modulation of the CD155/TIGIT axis was sufficient to promote immune evasion in autochthonous neoantigen-expressing PDAC. Finally, we demonstrate that the CD155/TIGIT axis is critical in maintaining immune evasion in PDAC and uncover a combination immunotherapy (TIGIT/PD-1 co-blockade plus CD40 agonism) that elicits profound anti-tumor responses in preclinical models, now poised for clinical evaluation.
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Evasión Inmune/inmunología , Inmunoterapia/métodos , Linfocitos Infiltrantes de Tumor/metabolismo , Neoplasias Pancreáticas/inmunología , Receptores Virales/inmunología , Animales , Humanos , Ratones , Neoplasias PancreáticasRESUMEN
A cell's phenotype and function are influenced by dynamic interactions with its microenvironment. To examine cellular spatiotemporal activity, we developed SPACECAT-Spatially PhotoActivatable Color Encoded Cell Address Tags-to annotate, track, and isolate cells while preserving viability. In SPACECAT, samples are stained with photocaged fluorescent molecules, and cells are labeled by uncaging those molecules with user-patterned near-UV light. SPACECAT offers single-cell precision and temporal stability across diverse cell and tissue types. Illustratively, we target crypt-like regions in patient-derived intestinal organoids to enrich for stem-like and actively mitotic cells, matching literature expectations. Moreover, we apply SPACECAT to ex vivo tissue sections from four healthy organs and an autochthonous lung tumor model. Lastly, we provide a computational framework to identify spatially-biased transcriptome patterns and enriched phenotypes. This minimally perturbative and broadly applicable method links cellular spatiotemporal and/or behavioral phenotypes with diverse downstream assays, enabling insights into the connections between tissue microenvironments and (dys)function.
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Rastreo Celular/psicología , Colorantes , Transcriptoma , Animales , Bioensayo , Citocinas , Femenino , Fluoresceínas , Colorantes Fluorescentes , Células HEK293 , Estado de Salud , Humanos , Neoplasias Pulmonares , Masculino , Ratones , Células Mieloides , Organoides , Fenotipo , Células Madre , Microambiente Tumoral , Rayos UltravioletaRESUMEN
Eukaryotic initiation factor 4A (eIF4A), the enzymatic core of the eIF4F complex essential for translation initiation, plays a key role in the oncogenic reprogramming of protein synthesis, and thus is a putative therapeutic target in cancer. As important component of its anticancer activity, inhibition of translation initiation can alleviate oncogenic activation of HSF1, a stress-inducible transcription factor that enables cancer cell growth and survival. Here, we show that primary acute myeloid leukemia (AML) cells exhibit the highest transcript levels of eIF4A1 compared to other cancer types. eIF4A inhibition by the potent and specific compound rohinitib (RHT) inactivated HSF1 in these cells, and exerted pronounced in vitro and in vivo anti-leukemia effects against progenitor and leukemia-initiating cells, especially those with FLT3-internal tandem duplication (ITD). In addition to its own anti-leukemic activity, genetic knockdown of HSF1 also sensitized FLT3-mutant AML cells to clinical FLT3 inhibitors, and this synergy was conserved in FLT3 double-mutant cells carrying both ITD and tyrosine kinase domain mutations. Consistently, the combination of RHT and FLT3 inhibitors was highly synergistic in primary FLT3-mutated AML cells. Our results provide a novel therapeutic rationale for co-targeting eIF4A and FLT3 to address the clinical challenge of treating FLT3-mutant AML.
Asunto(s)
Antineoplásicos/farmacología , Factor 4A Eucariótico de Iniciación/antagonistas & inhibidores , Factores de Transcripción del Choque Térmico/antagonistas & inhibidores , Leucemia Mieloide Aguda/tratamiento farmacológico , Animales , Humanos , Leucemia Mieloide Aguda/patología , Terapia Molecular DirigidaRESUMEN
Heat shock factor 1 (HSF1) is a cellular stress-protective transcription factor exploited by a wide range of cancers to drive proliferation, survival, invasion, and metastasis. Nuclear HSF1 abundance is a prognostic indicator for cancer severity, therapy resistance, and shortened patient survival. The HSF1 gene was amplified, and nuclear HSF1 abundance was markedly increased in prostate cancers and particularly in neuroendocrine prostate cancer (NEPC), for which there are no available treatment options. Despite genetic validation of HSF1 as a therapeutic target in a range of cancers, a direct and selective small-molecule HSF1 inhibitor has not been validated or developed for use in the clinic. We described the identification of a direct HSF1 inhibitor, Direct Targeted HSF1 InhiBitor (DTHIB), which physically engages HSF1 and selectively stimulates degradation of nuclear HSF1. DTHIB robustly inhibited the HSF1 cancer gene signature and prostate cancer cell proliferation. In addition, it potently attenuated tumor progression in four therapy-resistant prostate cancer animal models, including an NEPC model, where it caused profound tumor regression. This study reports the identification and validation of a direct HSF1 inhibitor and provides a path for the development of a small-molecule HSF1-targeted therapy for prostate cancers and other therapy-resistant cancers.
Asunto(s)
Factores de Transcripción del Choque Térmico/antagonistas & inhibidores , Neoplasias de la Próstata , Animales , Núcleo Celular/metabolismo , Humanos , Masculino , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/genéticaRESUMEN
The ability of cancer cells to cope with stressful conditions is critical for their survival, proliferation, and metastasis. The heat shock transcription factor 1 (HSF1) protects cells from stresses such as chemicals, radiation, and temperature. These properties of HSF1 are exploited by a broad spectrum of cancers, which exhibit high levels of nuclear, active HSF1. Functions for HSF1 in malignancy extend well beyond its central role in protein quality control. While HSF1 has been validated as a powerful target in cancers by genetic knockdown studies, HSF1 inhibitors reported to date have lacked sufficient specificity and potency for clinical evaluation. We review the roles of HSF1 in cancer, its potential as a prognostic indicator for cancer treatment, evaluate current HSF1 inhibitors and provide guidelines for the identification of selective HSF1 inhibitors as chemical probes and for clinical development.
Asunto(s)
Antineoplásicos/farmacología , Factores de Transcripción del Choque Térmico/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Animales , Antineoplásicos/uso terapéutico , Proliferación Celular/efectos de los fármacos , Factores de Transcripción del Choque Térmico/genética , Factores de Transcripción del Choque Térmico/metabolismo , Humanos , Terapia Molecular Dirigida , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Transducción de Señal/efectos de los fármacosRESUMEN
PURPOSE: Despite the accumulation of extensive genomic alterations, many cancers fail to be recognized as "foreign" and escape destruction by the host immune system. Immunotherapies designed to address this problem by directly stimulating immune effector cells have led to some remarkable clinical outcomes, but unfortunately, most cancers fail to respond, prompting the need to identify additional immunomodulatory treatment options.Experimental Design: We elucidated the effect of a novel treatment paradigm using sustained, low-dose HSP90 inhibition in vitro and in syngeneic mouse models using genetic and pharmacologic tools. Profiling of treatment-associated tumor cell antigens was performed using immunoprecipitation followed by peptide mass spectrometry. RESULTS: We show that sustained, low-level inhibition of HSP90 both amplifies and diversifies the antigenic repertoire presented by tumor cells on MHC-I molecules through an IFNγ-independent mechanism. In stark contrast, we find that acute, high-dose exposure to HSP90 inhibitors, the only approach studied in the clinic to date, is broadly immunosuppressive in cell culture and in patients with cancer. In mice, chronic non-heat shock-inducing HSP90 inhibition slowed progression of colon cancer implants, but only in syngeneic animals with intact immune function. Addition of a single dose of nonspecific immune adjuvant to the regimen dramatically increased efficacy, curing a subset of mice receiving combination therapy. CONCLUSIONS: These highly translatable observations support reconsideration of the most effective strategy for targeting HSP90 to treat cancers and suggest a practical approach to repurposing current orally bioavailable HSP90 inhibitors as a new immunotherapeutic strategy.See related commentary by Srivastava and Callahan, p. 6277.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias del Colon/tratamiento farmacológico , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Proteostasis/efectos de los fármacos , Animales , Presentación de Antígeno/efectos de los fármacos , Presentación de Antígeno/genética , Antígenos de Neoplasias/efectos de los fármacos , Antígenos de Neoplasias/inmunología , Línea Celular Tumoral , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Proteínas HSP90 de Choque Térmico/genética , Proteínas HSP90 de Choque Térmico/inmunología , Xenoinjertos , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Inmunoterapia/métodos , RatonesRESUMEN
Huntington's Disease (HD) is a neurodegenerative disease caused by poly-glutamine expansion in the Htt protein, resulting in Htt misfolding and cell death. Expression of the cellular protein folding and pro-survival machinery by heat shock transcription factor 1 (HSF1) ameliorates biochemical and neurobiological defects caused by protein misfolding. We report that HSF1 is degraded in cells and mice expressing mutant Htt, in medium spiny neurons derived from human HD iPSCs and in brain samples from patients with HD. Mutant Htt increases CK2α' kinase and Fbxw7 E3 ligase levels, phosphorylating HSF1 and promoting its proteasomal degradation. An HD mouse model heterozygous for CK2α' shows increased HSF1 and chaperone levels, maintenance of striatal excitatory synapses, clearance of Htt aggregates and preserves body mass compared with HD mice homozygous for CK2α'. These results reveal a pathway that could be modulated to prevent neuronal dysfunction and muscle wasting caused by protein misfolding in HD.
Asunto(s)
Encéfalo/metabolismo , Factores de Transcripción del Choque Térmico/metabolismo , Enfermedad de Huntington/metabolismo , Neuronas/metabolismo , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Células HEK293 , Factores de Transcripción del Choque Térmico/genética , Humanos , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Enfermedad de Huntington/genética , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Células PC12 , RatasRESUMEN
Heat-shock transcription factor (HSF) family members function in stress protection and in human diseases including proteopathies, neurodegeneration and cancer. The mechanisms that drive distinct post-translational modifications, cofactor recruitment and target-gene activation for specific HSF paralogs are unknown. We present crystal structures of the human HSF2 DNA-binding domain (DBD) bound to DNA, revealing an unprecedented view of HSFs that provides insights into their unique biology. The HSF2 DBD structures resolve a new C-terminal helix that directs wrapping of the coiled-coil domain around DNA, thereby exposing paralog-specific sequences of the DBD surface for differential post-translational modifications and cofactor interactions. We further demonstrate a direct interaction between HSF1 and HSF2 through their coiled-coil domains. Together, these features provide a new model for HSF structure as the basis for differential and combinatorial regulation, which influences the transcriptional response to cellular stress.
Asunto(s)
Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Cristalografía por Rayos X , ADN/química , ADN/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción del Choque Térmico , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , SumoilaciónRESUMEN
Heat shock transcription factor 1 (HSF1) is an evolutionarily conserved transcription factor that protects cells from protein-misfolding-induced stress and apoptosis. The mechanisms by which cytosolic protein misfolding leads to HSF1 activation have not been elucidated. Here, we demonstrate that HSF1 is directly regulated by TRiC/CCT, a central ATP-dependent chaperonin complex that folds cytosolic proteins. A small-molecule activator of HSF1, HSF1A, protects cells from stress-induced apoptosis, binds TRiC subunits in vivo and in vitro, and inhibits TRiC activity without perturbation of ATP hydrolysis. Genetic inactivation or depletion of the TRiC complex results in human HSF1 activation, and HSF1A inhibits the direct interaction between purified TRiC and HSF1 in vitro. These results demonstrate a direct regulatory interaction between the cytosolic chaperone machine and a critical transcription factor that protects cells from proteotoxicity, providing a mechanistic basis for signaling perturbations in protein folding to a stress-protective transcription factor.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Estrés del Retículo Endoplásmico , Complejos Multiproteicos/metabolismo , Factores de Transcripción/metabolismo , Animales , Apoptosis/efectos de los fármacos , Citoprotección/efectos de los fármacos , Estrés del Retículo Endoplásmico/efectos de los fármacos , Células HEK293 , Células HeLa , Factores de Transcripción del Choque Térmico , Humanos , Ratones , Células 3T3 NIH , Unión Proteica/efectos de los fármacos , Subunidades de Proteína/metabolismo , Ratas , Saccharomyces cerevisiae/metabolismo , Tunicamicina/farmacologíaRESUMEN
Inducible Hsp70 (Hsp70i) is overexpressed in a wide spectrum of human tumors, and its expression correlates with metastasis, poor outcomes, and resistance to chemotherapy in patients. Identification of small-molecule inhibitors selective for Hsp70i could provide new therapeutic tools for cancer treatment. In this work, we used fluorescence-linked enzyme chemoproteomic strategy (FLECS) to identify HS-72, an allosteric inhibitor selective for Hsp70i. HS-72 displays the hallmarks of Hsp70 inhibition in cells, promoting substrate protein degradation and growth inhibition. Importantly, HS-72 is selective for Hsp70i over the closely related constitutively active Hsc70. Studies with purified protein show HS-72 acts as an allosteric inhibitor, reducing ATP affinity. In vivo HS-72 is well-tolerated, showing bioavailability and efficacy, inhibiting tumor growth and promoting survival in a HER2+ model of breast cancer. The HS-72 scaffold is amenable to resynthesis and iteration, suggesting an ideal starting point for a new generation of anticancer therapeutics targeting Hsp70i.
Asunto(s)
Bencimidazoles/química , Bencimidazoles/farmacología , Proteínas HSP70 de Choque Térmico/antagonistas & inhibidores , Proteínas HSP70 de Choque Térmico/metabolismo , Ácidos Nipecóticos/química , Ácidos Nipecóticos/farmacología , Piperidinas/química , Piperidinas/farmacología , Regulación Alostérica/efectos de los fármacos , Animales , Bencimidazoles/metabolismo , Bencimidazoles/farmacocinética , Disponibilidad Biológica , Caspasas/metabolismo , Proliferación Celular/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Activación Enzimática/efectos de los fármacos , Células HEK293 , Proteínas HSP70 de Choque Térmico/química , Humanos , Ratones , Modelos Moleculares , Ácidos Nipecóticos/metabolismo , Ácidos Nipecóticos/farmacocinética , Permeabilidad , Piperidinas/metabolismo , Piperidinas/farmacocinética , Agregado de Proteínas/efectos de los fármacos , Estructura Terciaria de Proteína , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The heat shock transcription factor 1 (HSF1) activates expression of a variety of genes involved in cell survival, including protein chaperones, the protein degradation machinery, anti-apoptotic proteins, and transcription factors. Although HSF1 activation has been linked to amelioration of neurodegenerative disease, cancer cells exhibit a dependence on HSF1 for survival. Indeed, HSF1 drives a program of gene expression in cancer cells that is distinct from that activated in response to proteotoxic stress, and HSF1 DNA binding activity is elevated in cycling cells as compared with arrested cells. Active HSF1 homotrimerizes and binds to a DNA sequence consisting of inverted repeats of the pentameric sequence nGAAn, known as heat shock elements (HSEs). Recent comprehensive ChIP-seq experiments demonstrated that the architecture of HSEs is very diverse in the human genome, with deviations from the consensus sequence in the spacing, orientation, and extent of HSE repeats that could influence HSF1 DNA binding efficacy and the kinetics and magnitude of target gene expression. To understand the mechanisms that dictate binding specificity, HSF1 was purified as either a monomer or trimer and used to evaluate DNA-binding site preferences in vitro using fluorescence polarization and thermal denaturation profiling. These results were compared with quantitative chromatin immunoprecipitation assays in vivo. We demonstrate a role for specific orientations of extended HSE sequences in driving preferential HSF1 DNA binding to target loci in vivo. These studies provide a biochemical basis for understanding differential HSF1 target gene recognition and transcription in neurodegenerative disease and in cancer.