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The reaction of enamine compounds with the Togni reagent in the presence of CuI afforded ß-trifluoromethylated enamine intermediates, which were converted directly to biologically interesting trifluoromethylated 2H-azirines by an iodosobenzene (PhIO)-mediated intramolecular azirination in a one-pot process.
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BRAF V600E mutation is a driver mutation in the serrated pathway to colorectal cancers. BRAFV600E drives tumorigenesis through constitutive downstream extracellular signal-regulated kinase (ERK) activation, but high-intensity ERK activation can also trigger tumor suppression. Whether and how oncogenic ERK signaling can be intrinsically adjusted to a "just-right" level optimal for tumorigenesis remains undetermined. In this study, we found that FAK (Focal adhesion kinase) expression was reduced in BRAFV600E-mutant adenomas/polyps in mice and patients. In Vill-Cre;BRAFV600E/+;Fakfl/fl mice, Fak deletion maximized BRAFV600E's oncogenic activity and increased cecal tumor incidence to 100%. Mechanistically, our results showed that Fak loss, without jeopardizing BRAFV600E-induced ERK pathway transcriptional output, reduced EGFR (epidermal growth factor receptor)-dependent ERK phosphorylation. Reduction in ERK phosphorylation increased the level of Lgr4, promoting intestinal stemness and cecal tumor formation. Our findings show that a "just-right" ERK signaling optimal for BRAFV600E-induced cecal tumor formation can be achieved via Fak loss-mediated downregulation of ERK phosphorylation.
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Human papilloma virus (HPV) is an etiological factor of head and neck squamous cell carcinoma (HNSCC). To investigate the role of HPV antigen in anti-tumor immunity, we established mouse models by expressing HPV16 E6 and E7 in a SCC tumor cell line. We obtained two HPV antigen-expressing clones (C-225 and C-100) transplantable into C57BL/6 recipients. We found that C-225 elicited complete eradication in C57BL/6 mice (eradicated), whereas C-100 grew progressively (growing). We examined immune tumor microenvironment (TME) using flow cytometry and found that eradicated or growing tumors exhibited differential immune profiles that may influence the outcome of anti-tumor immunity. Surprisingly, the percentage of CD8 and CD4 tumor-infiltrating lymphocytes (TILs) was much higher in growing (C-100) than eradicated (C-225) tumor. However, the TILs upregulated PD-1 and LAG-3 more potently and exhibited impaired effector functions in growing tumor compared to their counterparts in eradicated tumor. C-225 TME is highly enriched with myeloid cells, especially polymorphonuclear (PMN) myeloid-derived suppressor cells (MDSC), whereas the percentage of M-MDSC and tumor-associated macrophages (TAMs) was much higher in C-100 TME, especially M2-TAMs (CD206+). The complete eradication of C-225 depended on CD8 T cells and elicited anti-tumor memory responses upon secondary tumor challenge. We employed DNA sequencing to identify differences in the T cell receptor of peripheral blood lymphocytes pre- and post-secondary tumor challenge. Lastly, C-225 and C-100 tumor lines harbored different somatic mutations. Overall, we uncovered differential immune TME that may underlie the divergent outcomes of anti-tumor immunity by establishing two SCC tumor lines, both of which express HPV16 E6 and E7 antigens. Our experimental models may provide a platform for pinpointing tumor-intrinsic versus host-intrinsic differences in orchestrating an immunosuppressive TME in HNSCCs and for identifying new targets that render tumor cells vulnerable to immune attack.
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Modelos Animales de Enfermedad , Linfocitos Infiltrantes de Tumor , Ratones Endogámicos C57BL , Proteínas Oncogénicas Virales , Proteínas E7 de Papillomavirus , Infecciones por Papillomavirus , Microambiente Tumoral , Animales , Microambiente Tumoral/inmunología , Ratones , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Línea Celular Tumoral , Proteínas Oncogénicas Virales/inmunología , Proteínas Oncogénicas Virales/genética , Proteínas E7 de Papillomavirus/inmunología , Proteínas E7 de Papillomavirus/genética , Infecciones por Papillomavirus/inmunología , Carcinoma de Células Escamosas de Cabeza y Cuello/inmunología , Carcinoma de Células Escamosas de Cabeza y Cuello/virología , Proteínas Represoras/genética , Proteína del Gen 3 de Activación de Linfocitos , Humanos , Progresión de la Enfermedad , Linfocitos T CD8-positivos/inmunología , Receptor de Muerte Celular Programada 1 , Femenino , Papillomavirus Humano 16/inmunología , Neoplasias de Cabeza y Cuello/inmunología , Neoplasias de Cabeza y Cuello/virologíaRESUMEN
BRAFV600E mutation is a driver mutation in the serrated pathway to colorectal cancers. BRAFV600E drives tumorigenesis through constitutive downstream extracellular signal-regulated kinase (ERK) activation, but high-intensity ERK activation can also trigger tumor suppression. Whether and how oncogenic ERK signaling can be intrinsically adjusted to a 'just-right' level optimal for tumorigenesis remains undetermined. In this study, we found that FAK (Focal adhesion kinase) expression was reduced in BRAFV600E-mutant adenomas/polyps in mice and patients. In Vil1-Cre;BRAFLSL-V600E/+;Ptk2fl/fl mice, Fak deletion maximized BRAFV600E's oncogenic activity and increased cecal tumor incidence to 100%. Mechanistically, our results showed that Fak loss, without jeopardizing BRAFV600E-induced ERK pathway transcriptional output, reduced EGFR (epidermal growth factor receptor)-dependent ERK phosphorylation. Reduction in ERK phosphorylation increased the level of Lgr4, promoting intestinal stemness and cecal tumor formation. Our findings show that a 'just-right' ERK signaling optimal for BRAFV600E-induced cecal tumor formation can be achieved via Fak loss-mediated downregulation of ERK phosphorylation.
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Neoplasias del Ciego , Quinasa 1 de Adhesión Focal , Proteínas Proto-Oncogénicas B-raf , Animales , Proteínas Proto-Oncogénicas B-raf/metabolismo , Proteínas Proto-Oncogénicas B-raf/genética , Fosforilación , Ratones , Humanos , Neoplasias del Ciego/metabolismo , Neoplasias del Ciego/genética , Neoplasias del Ciego/patología , Quinasa 1 de Adhesión Focal/metabolismo , Quinasa 1 de Adhesión Focal/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/genética , Sistema de Señalización de MAP Quinasas , Receptores ErbB/metabolismo , Receptores ErbB/genética , Carcinogénesis/genética , Carcinogénesis/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genética , MasculinoRESUMEN
PURPOSE: Cetuximab is a standard-of-care treatment for head and neck squamous cell carcinoma (HNSCC). Well-defined correlative markers of therapeutic responses are still lacking. Characterizing dynamic changes of T-cell receptor (TCR) repertoire in peripheral blood and tumor tissue may facilitate developing markers for cetuximab response in HNSCCs. EXPERIMENTAL DESIGN: We analyzed high-throughput TCRß sequencing data generated with ImmunoSEQ platform using peripheral blood mononuclear cells (PBMC) and tumor-infiltrating lymphocytes (TIL) from patients with HNSCC before and after cetuximab treatment (pre-/post-PBMC vs. pre-/post-TIL). Multiple analytic approaches were employed to normalize sequencing data. RESULTS: Normalized TCR richness was significantly lower in post-TIL than pre-TIL, suggesting that cetuximab reduced TCR diversity and promoted TCR expansion in TIL samples, regardless of response status. The magnitude of clonal expansion (defined as expansion rate) in top 20 TCR clonotypes was significantly higher in responder PBMC with or without normalization, and in responder TIL upon normalization, than nonresponder ones. Notably, the expanded top 20 or top 50 TCR clonotypes overlapped between PBMC and TIL samples, which occurred significantly more frequently in responders than nonresponders. CONCLUSIONS: Patients with cetuximab-treated HNSCC harbor dynamic changes of TCR repertoires correlative to therapeutic responses. The expansion rate of top TCR clonotypes in peripheral blood may serve as a minimally invasive, readily accessible, and feasible marker for predicting cetuximab responses in HNSCCs and beyond, and the expansion rate of top TCR clonotypes in TILs and their overlapping probability between PBMC and TIL may serve as additional predictive markers. Our study also highlights the importance of data normalization for TCR repertoire analysis.
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Neoplasias de Cabeza y Cuello , Linfocitos T , Humanos , Carcinoma de Células Escamosas de Cabeza y Cuello/tratamiento farmacológico , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Cetuximab/uso terapéutico , Receptores de Antígenos de Linfocitos T/genética , Linfocitos Infiltrantes de Tumor , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Neoplasias de Cabeza y Cuello/genéticaRESUMEN
Deletion of TRAF2 or TRAF3 in B cells prolongs their survival. However, it remains unknown whether deletion of such factors affects B cells' ability to tolerate DNA damage, which can be induced by chemotherapeutics and cause apoptosis. Genetic alterations of TRAF2 or TRAF3 are observed in subsets of human B-cell lymphomas and B cell-specific deletion of TRAF3 led to lymphoma development in aged mice. However, it remains unknown whether double deficiency of TRAF2 and TRAF3 accelerates B-cell lymphomagenesis. Here, we showed that B cell-specific TRAF2/3 double deficient (B-TRAF2/3-DKO) B cells were remarkably more resistant to DNA damage-induced apoptosis via upregulating cIAP2 and XIAP, which in turn attenuates caspase-3 activation. Mechanistically, resistance to DNA damage-induced apoptosis required NF-κB2, which effects by upregulating XIAP and cIAP2 transcription. B-TRAF2/3-DKO mice exhibited a shorter lifespan and succumbed to splenomegaly and lymphadenopathy. Unexpectedly, the incidence of B-cell lymphoma development in B-TRAF2/3-DKO mice was relatively rare (â¼10%). Sequencing B cell receptor repertoire of diseased B cells revealed that TRAF2/3 deficiency caused abnormal oligoclonal or clonal expansion of B cells. While a fraction of mutant B cells (25-43%) from aged diseased mice harbored recurrent chromosomal translocations, primary B cells isolated from young B-TRAF2/3-DKO mice had no detectable chromosomal alterations, suggesting that TRAF2/3 deficiency per se does not cause evident genomic instability in B cells. Chemo-resistant TRAF3-deficient B-cell lymphomas were sensitized to chemotherapeutic drugs by blocking IAP activity using IAP antagonist. We conclude that double deficiency of TRAF2 and TRAF3 does not accelerate B-cell lymphomagenesis. Our studies provide insight into mechanisms regulating DNA damage-induced apoptosis and may help develop effective therapies targeting mutant B-cell lymphomas using IAP antagonist.
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Linfoma de Células B , Linfoma , Humanos , Animales , Ratones , Anciano , Factor 2 Asociado a Receptor de TNF/genética , Factor 3 Asociado a Receptor de TNF/genética , Subunidad p52 de NF-kappa B , Apoptosis/genética , Daño del ADN , Linfoma de Células B/tratamiento farmacológico , Linfoma de Células B/genética , Proteína Inhibidora de la Apoptosis Ligada a XRESUMEN
Immune checkpoint inhibitors (ICIs) have revolutionized cancer treatment; however, the responses to ICI treatment are highly variable in different individuals and the underlying mechanisms remain poorly understood. Here, we employed a mouse squamous cell carcinoma (SCC) model where tumor-bearing recipients diverged into responders (R) versus non-responders (NR) upon anti-PD-L1 treatment. We performed in-depth TCRß sequencing with immunoSEQ platform to delineate the differences in CD8 tumor-infiltrating lymphocytes (TILs). We found that R and NR CD8 TILs both exhibited evidence of clonal expansion, suggesting activation regardless of response status. We detected no differences in clonal expansion or clonal diversity indexes between R vs. NR. However, the top expanded (>1%) TCRß clonotypes appeared to be mutually exclusive between R and NR CD8 TILs, showing a preferential expansion of distinct TCRß clonotypes in response to the same SCC tumor in R vs. NR. Notably, the mutual exclusivity of TCR clonotypes in R vs. NR was only observed when top TCRß clonotypes were counted, because such top-expanded clonotypes are present in the opposite outcome group at a much lower frequency. Many TCRß sequences were detected in only one recipient at a high frequency, implicating highly individualized anti-tumor immune responses. We conclude that differences in the clonal frequency of top TCR clonotypes between R and NR CD8 TILs may be one of the factors underlying differential anti-PD-L1 responses. This notion may offer a novel explanation for variable ICI responses in different individuals, which may substantially impact the development of new strategies for personalized cancer immunotherapy.
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Linfocitos T CD8-positivos , Inmunoterapia , Animales , Ratones , Receptores de Antígenos de Linfocitos TRESUMEN
Differential responses to immune checkpoint inhibitors (ICI) may be attributed to tumor-intrinsic factors or environmental cues; however, these mechanisms cannot fully explain the variable ICI responses in different individuals. Here, we investigate the potential contribution of immunological heterogeneity with a focus on differences in T-cell receptor (TCR) repertoire to ICI responses, which has not been defined previously. To reveal additional factors underlying heterogeneous responses to ICI, we employed a squamous cell carcinoma (SCC) mouse model in which tumor-bearing recipients unambiguously diverged into responders (R) or non-responders (NR) upon anti-PD-L1 treatment. Treatment efficacy absolutely required CD8 T-cells and correlated positively with effector functions of CD8 tumor-infiltrating lymphocytes (TILs). We showed that TCR repertoires exhibited a similar magnitude of clonal expansion in R vs. NR CD8 TILs. However, the top expanded TCR clonotypes appeared to be mutually exclusive between R and NR CD8 TILs, which also occurred in a recipient-specific manner, demonstrating preferential expansion of distinct TCR clonotypes against the same SCC tumor. Unexpectedly, R vs. NR CD8 TILs reached all activation clusters and did not exhibit substantial global differences in transcriptomes. By linking single-cell transcriptomic data with unique TCR clonotypes, CD8 TILs harboring top TCR clonotypes were found to occupy distinct activation clusters and upregulate genes favoring anti-tumor immunity to different extents in R vs. NR. We conclude that stochastic differences in CD8 TIL TCR repertoire and distinct activation states of top TCR clonotypes may contribute to differential anti-PD-L1 responses. Our study suggests that host-intrinsic immunological heterogeneity may offer a new explanation for differential ICI responses in different individuals, which could impact on strategies for personalized cancer immunotherapy.
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Carcinoma de Células Escamosas , Linfocitos Infiltrantes de Tumor , Ratones , Animales , Linfocitos T CD8-positivos , Activación de Linfocitos , Receptores de Antígenos de Linfocitos T/genéticaRESUMEN
BACKGROUND: While immune checkpoint inhibitors (ICI) were approved for head and neck squamous cell carcinomas (HNSCCs), the response rate remains relatively low. Mechanisms underlying ICI unresponsiveness versus sensitivity are not fully understood. METHOD: To better delineate differential responses to ICI treatment, we employed mouse SCC models, termed KPPA tumors that were caused by deleting p53 and hyperactivating PIK3CA, two most frequently mutated genes in human HNSCCs. We transplanted two KPPA tumor lines (TAb2 versus TCh3) into C57BL/6 recipients and examined the immune tumor microenvironment using flow cytometry. Furthermore, we employed single-cell RNA sequencing to identify the difference in tumor infiltrating lymphocytes (TILs). RESULTS: We found that different KPPA tumors exhibited heterogeneous immune profiles pre-existing treatment that dictated their sensitivity or unresponsiveness to anti-PD-L1. Unresponsive TAb2 tumors were highly enriched with functional tumor-associated macrophages (TAMs), especially M2-TAMs. In contrast, sensitive TCh3 tumors contained more CD8 TILs with better effector functions. TAb2 tumor cells drastically expanded F4/80+ TAMs from bone marrow precursors, requiring CSF1 and VEGF. Consistently, a higher combined expression of VEGF-C and CSF1 predicts worse survival in PIK3CAAmp/TP53Mutated HNSCC patients. Unresponsive TAb2 tumors upregulated distinct signaling pathways that correlate with aggressive tumor phenotypes. While anti-PD-L1 did not affect the TME of TAb2 tumors, it significantly increased the number of CD8 TILs in TCh3 tumors. CONCLUSIONS: We uncovered tumor-intrinsic differences that may underlie the differential responses to ICI by establishing and employing two SCC tumor lines, TAb2 vs. TCh3, both of which harbor TP53 deletion and PIK3CA hyperactivation. Our study indicates the limitation of stratifying cancers according to their genetic alterations and suggests that evaluating HNSCC tumor-intrinsic cues along with immune profiles in the TME may help better predict ICI responses. Our experimental models may provide a platform for pinpointing tumor-intrinsic differences underlying an immunosuppressive TME in HNSCCs and for testing combined immunotherapies targeting either tumor-specific or TAM-specific players to improve ICI efficacy.
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Carcinoma de Células Escamosas , Neoplasias de Cabeza y Cuello , Animales , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Humanos , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Ratones , Ratones Endogámicos C57BL , Oncogenes , Microambiente TumoralRESUMEN
An exclusive thiophene-fused polycyclic π-conjugated 2-arylbenzo[4,5]thieno[2,3-d]thiazole skeleton was constructed via a one-pot CuCl-mediated three-component reaction, using 2-(2-bromophenyl)acetonitrile and aromatic aldehydes as substrates and elemental sulfur as sulfur source in the presence of K2CO3 and 1,10-phen in DMSO. A plausible reaction mechanism was proposed, which involved formation of benzo[b]thiophen-2-amines through cyclization of 2-bromophenyl acetonitrile and sulfur, and subsequent intramolecular condensation/dehydrogenation with aromatic aldehydes.