RESUMO
The majority of clinically approved drugs target proteins that are secreted or cell surface bound. However, further advances in this area have been hindered by the challenging nature of receptor deorphanization, as there are still many secreted and cell-bound proteins with unknown binding partners. Here, we developed an advanced screening platform that combines CRISPR-CAS9 guide-mediated gene activation (CRISPRa) and high-avidity bead-based selection. The CRISPRa platform incorporates serial enrichment and flow cytometry-based monitoring, resulting in substantially improved screening sensitivity for well-known yet weak interactions of the checkpoint inhibitor family. Our approach has successfully revealed that siglec-4 exerts regulatory control over T cell activation through a low affinity trans-interaction with the costimulatory receptor 4-1BB. Our highly efficient screening platform holds great promise for identifying extracellular interactions of uncharacterized receptor-ligand partners, which is essential to develop next-generation therapeutics, including additional immune checkpoint inhibitors.
Assuntos
Sistemas CRISPR-Cas , Proteínas de Membrana , Ligantes , Proteínas de Membrana/genética , Ativação TranscricionalRESUMO
CD276/B7-H3 represents a promising target for cancer therapy based on widespread overexpression in both cancer cells and tumor-associated stroma. In previous preclinical studies, CD276 antibody-drug conjugates (ADCs) exploiting a talirine-type pyrrolobenzodiazepine (PBD) payload showed potent activity against various solid tumors but with a narrow therapeutic index and dosing regimen higher than that tolerated in clinical trials using other antibody-talirine conjugates. Here, we describe the development of a modified talirine PBD-based fully human CD276 ADC, called m276-SL-PBD, that is cross-species (human/mouse) reactive and can eradicate large 500-1,000-mm3 triple-negative breast cancer xenografts at doses 10- to 40-fold lower than the maximum tolerated dose. By combining CD276 targeting with judicious genetic and chemical ADC engineering, improved ADC purification, and payload sensitivity screening, these studies demonstrate that the therapeutic index of ADCs can be substantially increased, providing an advanced ADC development platform for potent and selective targeting of multiple solid tumor types.
Assuntos
Imunoconjugados , Neoplasias , Humanos , Camundongos , Animais , Imunoconjugados/farmacologia , Linhagem Celular Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto , Anticorpos Monoclonais Humanizados , Fatores de Transcrição , Neoplasias/tratamento farmacológico , Antígenos B7RESUMO
Collagen I, the most abundant protein in humans, is ubiquitous in solid tumors where it provides a rich source of exploitable metabolic fuel for cancer cells. While tumor cells were unable to exploit collagen directly, here we show they can usurp metabolic byproducts of collagen-consuming tumor-associated stroma. Using genetically engineered mouse models, we discovered that solid tumor growth depends upon collagen binding and uptake mediated by the TEM8/ANTXR1 cell surface protein in tumor-associated stroma. Tumor-associated stromal cells processed collagen into glutamine, which was then released and internalized by cancer cells. Under chronic nutrient starvation, a condition driven by the high metabolic demand of tumors, cancer cells exploited glutamine to survive, an effect that could be reversed by blocking collagen uptake with TEM8 neutralizing antibodies. These studies reveal that cancer cells exploit collagen-consuming stromal cells for survival, exposing an important vulnerability across solid tumors with implications for developing improved anticancer therapy.
Assuntos
Imunoconjugados , Neoplasias , Humanos , Camundongos , Animais , Sobrevivência Celular , Glutamina , Colágeno/metabolismo , Proteínas dos Microfilamentos , Receptores de Superfície CelularRESUMO
Although nonmalignant stromal cells facilitate tumor growth and can occupy up to 90% of a solid tumor mass, better strategies to exploit these cells for improved cancer therapy are needed. Here, we describe a potent MMAE-linked antibody-drug conjugate (ADC) targeting tumor endothelial marker 8 (TEM8, also known as ANTXR1), a highly conserved transmembrane receptor broadly overexpressed on cancer-associated fibroblasts, endothelium, and pericytes. Anti-TEM8 ADC elicited potent anticancer activity through an unexpected killing mechanism we term DAaRTS (drug activation and release through stroma), whereby the tumor microenvironment localizes active drug at the tumor site. Following capture of ADC prodrug from the circulation, tumor-associated stromal cells release active MMAE free drug, killing nearby proliferating tumor cells in a target-independent manner. In preclinical studies, ADC treatment was well tolerated and induced regression and often eradication of multiple solid tumor types, blocked metastatic growth, and prolonged overall survival. By exploiting TEM8+ tumor stroma for targeted drug activation, these studies reveal a drug delivery strategy with potential to augment therapies against multiple cancer types.
Assuntos
Imunoconjugados/farmacologia , Proteínas de Neoplasias/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Receptores de Superfície Celular/antagonistas & inibidores , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Biomarcadores Tumorais/antagonistas & inibidores , Biomarcadores Tumorais/deficiência , Biomarcadores Tumorais/genética , Brentuximab Vedotin , Linhagem Celular Tumoral , Feminino , Humanos , Imunoconjugados/farmacocinética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Nus , Camundongos SCID , Proteínas dos Microfilamentos , Neoplasias/metabolismo , Receptores de Peptídeos/antagonistas & inibidores , Receptores de Peptídeos/deficiência , Receptores de Peptídeos/genética , Células Estromais/efeitos dos fármacos , Microambiente Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
[This corrects the article DOI: 10.1186/s13578-018-0204-8.].
RESUMO
Promyelocytic leukemia protein (PML) was originally identified as a fusion partner of retinoic acid receptor alpha in acute promyelocytic leukemia patients with the (15;17) chromosomal translocation, giving rise to PML-RARα and RARα-PML fusion proteins. A body of evidence indicated that PML possesses tumor suppressing activity by regulating apoptosis, cell cycle, senescence and DNA damage responses. PML is enriched in discrete nuclear substructures in mammalian cells with 0.2-1 µm diameter in size, referred to as alternately Kremer bodies, nuclear domain 10, PML oncogenic domains or PML nuclear bodies (NBs). Dysregulation of PML NB formation results in altered transcriptional regulation, protein modification, apoptosis and cellular senescence. In addition to PML NBs, PML is also present in nucleoplasm and cytoplasmic compartments, including the endoplasmic reticulum and mitochondria-associated membranes. The role of PML in tumor suppression has been extensively studied but increasing evidence indicates that PML also plays versatile roles in stem cell renewal, metabolism, inflammatory responses, neural function, mammary development and angiogenesis. In this review, we will briefly describe the known PML regulation and function and include new findings.
RESUMO
IFNs are effective in inhibiting angiogenesis in preclinical models and in treating several angioproliferative disorders. However, the detailed mechanisms of IFNα-mediated anti-angiogenesis are not completely understood. Stat1/2/3 and PML are IFNα downstream effectors and are pivotal regulators of angiogenesis. Here, we investigated PML's role in the regulation of Stat1/2/3 activity. In Pml knock-out (KO) mice, ablation of Pml largely reduces IFNα angiostatic ability in Matrigel plug assays. This suggested an essential role for PML in IFNα's anti-angiogenic function. We also demonstrated that PML shared a large cohort of regulatory genes with Stat1 and Stat3, indicating an important role of PML in regulating Stat1 and Stat3 activity. Using molecular tools and primary endothelial cells, we demonstrated that PML positively regulates Stat1 and Stat2 isgylation, a ubiquitination-like protein modification. Accordingly, manipulation of the isgylation system by knocking down USP18 altered IFNα-PML axis-mediated inhibition of endothelial cell migration and network formation. Furthermore, PML promotes turnover of nuclear Stat3, and knockdown of PML mitigates the effect of LLL12, a selective Stat3 inhibitor, on IFNα-mediated anti-angiogenic activity. Taken together, we elucidated an unappreciated mechanism in which PML, an IFNα-inducible effector, possess potent angiostatic activity, doing so in part by forming a positive feedforward loop with Stat1/2 and a negative feedback loop with Stat3. The interplay between PML, Stat1/Stat2, and Stat3 contributes to IFNα-mediated inhibition of angiogenesis, and disruption of this network results in aberrant IFNα signaling and altered angiostatic activity.
Assuntos
Endotélio Vascular/metabolismo , Interferon-alfa/metabolismo , Neovascularização Patológica/prevenção & controle , Proteína da Leucemia Promielocítica/metabolismo , Fator de Transcrição STAT1/agonistas , Fator de Transcrição STAT2/agonistas , Fator de Transcrição STAT3/antagonistas & inibidores , Animais , Linhagem Celular , Células Cultivadas , Endopeptidases/química , Endopeptidases/genética , Endopeptidases/metabolismo , Endotélio Vascular/citologia , Endotélio Vascular/patologia , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Interferon-alfa/genética , Camundongos Knockout , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Neovascularização Fisiológica , Proteína da Leucemia Promielocítica/antagonistas & inibidores , Proteína da Leucemia Promielocítica/genética , Processamento de Proteína Pós-Traducional , Interferência de RNA , Proteínas Recombinantes/metabolismo , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT2/genética , Fator de Transcrição STAT2/metabolismo , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Ubiquitina Tiolesterase/antagonistas & inibidores , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismoRESUMO
Glomerular podocytes are highly specialized terminally differentiated cells that act as a filtration barrier in the kidney. Mutations in the actin-binding protein, α-actinin 4 (ACTN4), are linked to focal segmental glomerulosclerosis (FSGS), a chronic kidney disease characterized by proteinuria. Aberrant activation of NF-κB pathway in podocytes is implicated in glomerular diseases including proteinuria. We demonstrate here that stable knockdown of ACTN4 in podocytes significantly reduces TNFα-mediated induction of NF-κB target genes, including IL-1ß and NPHS1, and activation of an NF-κB-driven reporter without interfering with p65 nuclear translocation. Overexpression of ACTN4 and an actin binding-defective variant increases the reporter activity. In contrast, an FSGS-linked ACTN4 mutant, K255E, which has increased actin binding activity and is predominantly cytoplasmic, fails to potentiate NF-κB activity. Mechanistically, IκBα blocks the association of ACTN4 and p65 in the cytosol. In response to TNFα, both NF-κB subunits p65 and p50 translocate to the nucleus, where they bind and recruit ACTN4 to their targeted promoters, IL-1ß and IL-8. Taken together, our data identify ACTN4 as a novel coactivator for NF-κB transcription factors in podocytes. Importantly, this nuclear function of ACTN4 is independent of its actin binding activity in the cytoplasm.
Assuntos
Actinina/genética , NF-kappa B/genética , Podócitos/metabolismo , Transcrição Gênica , Actinina/antagonistas & inibidores , Actinina/metabolismo , Actinas/genética , Actinas/metabolismo , Animais , Linhagem Celular Transformada , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Podócitos/citologia , Ligação Proteica , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de SinaisRESUMO
Cytokine modulation of the endothelium is considered an important contributor to the inflammation response. TNFα is an early response gene during the initiation of inflammation. However, the detailed mechanism by which TNFα induces proinflammatory gene expression is not completely understood. In this report, we demonstrate that silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) represses the expression of a subset of TNFα target genes in human umbilical vein endothelial cells. Upon TNFα stimulation, we observed an increase in the E3 ubiquitin ligase ß-TrCP1 and a decrease in SMRT protein levels. We show that ß-TrCP1 interacts with SMRT in a phosphorylation-independent manner and cooperates with the E2 ubiquitin-conjugating enzyme E2D2 to promote ubiquitination-dependent SMRT degradation. Knockdown of ß-TrCP1 increases SMRT protein accumulation, increases SMRT association with its targeted promoters, and decreases SMRT target gene expression. Taken together, our results support a model in which TNFα-induced ß-TrCP1 accumulation promotes SMRT degradation and the subsequent induction of proinflammatory gene expression.
Assuntos
Regulação da Expressão Gênica/fisiologia , Modelos Biológicos , Correpressor 2 de Receptor Nuclear/metabolismo , Proteólise , Fator de Necrose Tumoral alfa/biossíntese , Proteínas Contendo Repetições de beta-Transducina/metabolismo , Células HEK293 , Células HeLa , Células Endoteliais da Veia Umbilical Humana , Humanos , Correpressor 2 de Receptor Nuclear/genética , Fosforilação/fisiologia , Fator de Necrose Tumoral alfa/genética , Ubiquitina/genética , Ubiquitina/metabolismo , Proteínas Contendo Repetições de beta-Transducina/genéticaRESUMO
Alpha-actinins (ACTNs) were originally identified as cytoskeletal proteins which cross-link filamentous actin to establish cytoskeletal architect that protects cells from mechanical stress and controls cell movement. Notably, unlike other ACTNs, alpha-actinin 4 (ACTN4) displays unique characteristics in signaling transduction, nuclear translocation, and gene expression regulation. Initial reports indicated that ACTN4 is part of the breast cancer cell motile apparatus and is highly expressed in the nucleus. These results imply that ACTN4 plays a role in breast cancer tumorigenesis. While several observations in breast cancer and other cancers support this hypothesis, little direct evidence links the tumorigenic phenotype with ACTN4-mediated pathological mechanisms. Recently, several studies have demonstrated that in addition to its role in coordinating cytoskeleton, ACTN4 interacts with signaling mediators, chromatin remodeling factors, and transcription factors including nuclear receptors. Thus, ACTN4 functions as a versatile promoter for breast cancer tumorigenesis and appears to be an ideal drug target for future therapeutic development.