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1.
Nat Commun ; 15(1): 3581, 2024 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-38678024

RESUMO

Immune checkpoint blockade therapy aims to activate the immune system to eliminate cancer cells. However, clinical benefits are only recorded in a subset of patients. Here, we leverage genome-wide CRISPR/Cas9 screens in a Tumor-Immune co-Culture System focusing on triple-negative breast cancer (TNBC). We reveal that NEDD8 loss in cancer cells causes a vulnerability to nivolumab (anti-PD-1). Genetic deletion of NEDD8 only delays cell division initially but cell proliferation is unaffected after recovery. Since the NEDD8 gene is commonly essential, we validate this observation with additional CRISPR screens and uncover enhanced immunogenicity in NEDD8 deficient cells using proteomics. In female immunocompetent mice, PD-1 blockade lacks efficacy against established EO771 breast cancer tumors. In contrast, we observe tumor regression mediated by CD8+ T cells against Nedd8 deficient EO771 tumors after PD-1 blockade. In essence, we provide evidence that NEDD8 is conditionally essential in TNBC and presents as a synergistic drug target for PD-1/L1 blockade therapy.


Assuntos
Inibidores de Checkpoint Imunológico , Proteína NEDD8 , Neoplasias de Mama Triplo Negativas , Animais , Feminino , Humanos , Camundongos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sistemas CRISPR-Cas , Inibidores de Checkpoint Imunológico/farmacologia , Proteína NEDD8/metabolismo , Proteína NEDD8/genética , Neoplasias de Mama Triplo Negativas/imunologia , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia
2.
J Clin Invest ; 133(7)2023 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-36757800

RESUMO

Inflammatory mediators released by cancer cells promote the induction of immune suppression and tolerance in myeloid cells. IL-1 receptor-associated kinase-3 (IRAK3) is a pseudokinase that inhibits IL-1/TLR signaling, but its role in patients treated with immune checkpoint blockade (ICB) therapy remains unclear. Using RNA-Seq data from the IMvigor210 trial, we found that tumors with high IRAK3 expressions showed enriched antiinflammatory pathways and worse clinical response to ICB therapy. Upon IRAK3 protein deletion with CRISPR/Cas9, primary human monocytes displayed altered global protein expression and phosphorylation in quantitative proteomics and released more proinflammatory cytokines in response to stimulation. Bone marrow-derived macrophages from an IRAK3 CRISPR KO mouse model demonstrated a proinflammatory phenotype and enhanced sensitivity to TLR agonists compared with WT cells. IRAK3 deficiency delayed the growth of carcinogen-induced and oncogene-driven murine cancer cells and induced enhanced activation in myeloid cells and T cells. Upon ICB treatment, IRAK3-KO mice showed enrichment of TCF1+PD-1+ stem-like memory CD8+ T cells and resulted in superior growth inhibition of immunologically cold tumors in vivo. Altogether, our study demonstrated what we believe to be a novel cancer-driven immune tolerance program controlled by IRAK3 in humans and mice and proposed its suitability as an immunotherapy target.


Assuntos
Quinases Associadas a Receptores de Interleucina-1 , Neoplasias , Humanos , Animais , Camundongos , Quinases Associadas a Receptores de Interleucina-1/genética , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Células Mieloides/patologia , Macrófagos/metabolismo , Imunoterapia , Neoplasias/genética , Neoplasias/terapia , Microambiente Tumoral
3.
Mol Biotechnol ; 59(9-10): 407-424, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28801725

RESUMO

Human rhinovirus (HRV) 3C protease is widely used in recombinant protein production for various applications such as biochemical characterization and structural biology projects to separate recombinant fusion proteins from their affinity tags in order to prevent interference between these tags and the target proteins. Herein, we report the optimization of expression and purification conditions of glutathione S-transferase (GST)-tagged HRV 3C protease by statistically designed experiments. Soluble expression of GST-HRV 3C protease was initially optimized by response surface methodology (RSM), and a 5.5-fold increase in enzyme yield was achieved. Subsequently, we developed a new incomplete factorial (IF) design that examines four variables (bacterial strain, expression temperature, induction time, and inducer concentration) in a single experiment. The new design called Incomplete Factorial-Strain/Temperature/Time/Inducer (IF-STTI) was validated using three GST-tagged proteins. In all cases, IF-STTI resulted in only 10% lower expression yields than those obtained by RSM. Purification of GST-HRV 3C was optimized by an IF design that examines simultaneously the effect of the amount of resin, incubation time of cell lysate with resin, and glycerol and DTT concentration in buffers, and a further 15% increase in protease recovery was achieved. Purified GST-HRV 3C protease was active at both 4 and 25 °C in a variety of buffers.


Assuntos
Cisteína Endopeptidases/genética , Proteínas Recombinantes de Fusão/genética , Rhinovirus/genética , Proteínas Virais/genética , Proteases Virais 3C , Cisteína Endopeptidases/química , Cisteína Endopeptidases/isolamento & purificação , Regulação Viral da Expressão Gênica , Glutationa Transferase/química , Glutationa Transferase/genética , Humanos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Rhinovirus/enzimologia , Proteínas Virais/química , Proteínas Virais/isolamento & purificação
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