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1.
J Immunother Cancer ; 10(3)2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35264439

RESUMO

BACKGROUND: Genetically engineered T-cell immunotherapies for adoptive cell transfer (ACT) have emerged as a promising form of cancer treatment, but many of these patients develop recurrent disease. Furthermore, delineating mechanisms of resistance may be challenging since the analysis of bulk tumor profiling can be complicated by spatial heterogeneity. METHODS: Tumor samples were collected from a patient with synovial sarcoma who developed acquired resistance to ACT targeting NY-ESO-1. Biopsies (primary, progressive metastasis, and recurrence) were subjected to bulk tumor DNA and RNA sequencing, as well as high-dimensional spatial profiling of RNA and protein targets. Untreated and progressive lesions were compared with identified patterns associated with acquired resistance to ACT. RESULTS: Gene expression patterns due to immune activity and infiltration were diluted in bulk tumor sequencing. The metastasis was enriched for tumor regions with increased CTNNB1 (encoding beta-catenin), which were negatively associated with the expression of T-cell surface proteins and antigen presentation machinery. Spatial profiling was most highly concordant with bulk sequencing in the lesions with decreased spatial heterogeneity. CONCLUSIONS: Complementary use of bulk and spatial profiling enables more accurate interrogation of tumor specimens, particularly to address complex questions regarding immunotherapeutic mechanisms. Our study uses this approach to demonstrate a mechanism of T-cell exclusion and resistance to cellular immunotherapy in synovial sarcoma.


Assuntos
Sarcoma Sinovial , Antígenos de Neoplasias , Terapia Baseada em Transplante de Células e Tecidos , Humanos , Sarcoma Sinovial/genética , Sarcoma Sinovial/terapia , Linfócitos T , Via de Sinalização Wnt
2.
Cell Rep ; 36(4): 109443, 2021 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-34320363

RESUMO

Metastasis is a complex and poorly understood process. In pancreatic cancer, loss of the transforming growth factor (TGF)-ß/BMP effector SMAD4 is correlated with changes in altered histopathological transitions, metastatic disease, and poor prognosis. In this study, we use isogenic cancer cell lines to identify SMAD4 regulated genes that contribute to the development of metastatic colonization. We perform an in vivo screen identifying FOSL1 as both a SMAD4 target and sufficient to drive colonization to the lung. The targeting of these genes early in treatment may provide a therapeutic benefit.


Assuntos
Regulação Neoplásica da Expressão Gênica , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas c-fos/genética , Proteína Smad4/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/patologia , Animais , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Elementos Facilitadores Genéticos/genética , Humanos , Camundongos , Metástase Neoplásica , Proteínas Proto-Oncogênicas c-fos/metabolismo , Neoplasias Pancreáticas
3.
Cell Rep ; 29(1): 118-134.e8, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31577942

RESUMO

The mitogen-activated protein kinase (MAPK) pathway is a critical effector of oncogenic RAS signaling, and MAPK pathway inhibition may be an effective combination treatment strategy. We performed genome-scale loss-of-function CRISPR-Cas9 screens in the presence of a MEK1/2 inhibitor (MEKi) in KRAS-mutant pancreatic and lung cancer cell lines and identified genes that cooperate with MEK inhibition. While we observed heterogeneity in genetic modifiers of MEKi sensitivity across cell lines, several recurrent classes of synthetic lethal vulnerabilities emerged at the pathway level. Multiple members of receptor tyrosine kinase (RTK)-RAS-MAPK pathways scored as sensitizers to MEKi. In particular, we demonstrate that knockout, suppression, or degradation of SHOC2, a positive regulator of MAPK signaling, specifically cooperated with MEK inhibition to impair proliferation in RAS-driven cancer cells. The depletion of SHOC2 disrupted survival pathways triggered by feedback RTK signaling in response to MEK inhibition. Thus, these findings nominate SHOC2 as a potential target for combination therapy.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neoplasias/metabolismo , Proteínas ras/metabolismo , Células A549 , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Células HCT116 , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Camundongos Pelados , Camundongos SCID , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia
4.
Artigo em Inglês | MEDLINE | ID: mdl-30038041

RESUMO

The cell wall of Gram-positive bacteria contains abundant surface-exposed carbohydrate structures that are highly conserved. While these properties make surface carbohydrates ideal targets for immunotherapy, carbohydrates elicit a poor immune response that results primarily in low-affinity IgM antibodies. In a previous publication, we introduced the lysibody approach to address this shortcoming. Lysibodies are engineered molecules that combine a high-affinity carbohydrate-binding domain of bacterial or bacteriophage origin and an Fc effector portion of a human IgG antibody, thus directing effective immunity to conserved bacterial surface carbohydrates. Here, we describe the first example of a lysibody containing the binding domain from a bacteriocin, lysostaphin. We also describe the creation of five lysibodies with binding domains derived from phage lysins, directed against Staphylococcus aureus The lysostaphin and LysK lysibodies showed the most promise and were further characterized. Both lysibodies bound a range of clinically important staphylococcal strains, fixed complement on the staphylococcal surface, and induced phagocytosis of S. aureus by macrophages and human neutrophils. The lysostaphin lysibody had superior in vitro activity compared to that of the LysK lysibody, as well as that of the previously characterized ClyS lysibody, and it effectively protected mice in a kidney abscess/bacteremia model. These results further demonstrate that the lysibody approach is a reproducible means of creating antibacterial antibodies that cannot be produced by conventional means. Lysibodies therefore are a promising solution for opsonic antibodies that may be used passively to both treat and prevent infection by drug-resistant pathogens.


Assuntos
Antibacterianos/farmacologia , Lisostafina/farmacologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/enzimologia , Animais , Camundongos , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Proteínas Opsonizantes/metabolismo
5.
Proc Natl Acad Sci U S A ; 114(18): 4781-4786, 2017 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-28428342

RESUMO

The cell wall of Gram-positive bacteria contains abundant surface-exposed carbohydrate molecules that are highly conserved within and often across species. The potential therapeutic usefulness of high-affinity antibodies to cell wall carbohydrates is unquestioned, however obtaining such antibodies is challenging due to the poor overall immunogenicity of these bacterial targets. Autolysins and phage lysins are peptidoglycan hydrolases, enzymes that have evolved over a billion years to degrade bacterial cell wall. Such wall hydrolases are modular enzymes, composed of discrete domains for high-affinity binding to cell wall carbohydrates and cleavage activity. In this study, we demonstrate that binding domains from autolysins and lysins can be fused to the Fc region of human IgG, creating a fully functional homodimer (or "lysibody") with high-affinity binding and specificity for carbohydrate determinants on the bacterial surface. Furthermore, we demonstrate that this process is reproducible with three different binding domains specific to methicillin-resistant Staphylococcus aureus (MRSA). Cell-bound lysibodies induced the fixation of complement on the bacterial surface, promoted phagocytosis by macrophages and neutrophils, and protected mice from MRSA infection in two model systems. The lysibody approach could be used to target a range of difficult-to-treat pathogenic bacteria, given that cell wall hydrolases are ubiquitous in nature.


Assuntos
Parede Celular/metabolismo , Fragmentos Fc das Imunoglobulinas/farmacologia , N-Acetil-Muramil-L-Alanina Amidase/farmacologia , Fagocitose/efeitos dos fármacos , Polissacarídeos Bacterianos/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Staphylococcus aureus/metabolismo , Células HL-60 , Humanos , Fragmentos Fc das Imunoglobulinas/genética , N-Acetil-Muramil-L-Alanina Amidase/genética , Polissacarídeos Bacterianos/antagonistas & inibidores , Proteínas Recombinantes de Fusão/genética
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