RESUMO
Pharmacogenomics is a rapidly growing field with the goal of providing personalized care to every patient. Previously, we developed the Computational Analysis of Novel Drug Opportunities (CANDO) platform for multiscale therapeutic discovery to screen optimal compounds for any indication/disease by performing analytics on their interactions using large protein libraries. We implemented a comprehensive precision medicine drug discovery pipeline within the CANDO platform to determine which drugs are most likely to be effective against mutant phenotypes of non-small cell lung cancer (NSCLC) based on the supposition that drugs with similar interaction profiles (or signatures) will have similar behavior and therefore show synergistic effects. CANDO predicted that osimertinib, an EGFR inhibitor, is most likely to synergize with four KRAS inhibitors.Validation studies with cellular toxicity assays confirmed that osimertinib in combination with ARS-1620, a KRAS G12C inhibitor, and BAY-293, a pan-KRAS inhibitor, showed a synergistic effect on decreasing cellular proliferation by acting on mutant KRAS. Gene expression studies revealed that MAPK expression is strongly correlated with decreased cellular proliferation following treatment with KRAS inhibitor BAY-293, but not treatment with ARS-1620 or osimertinib. These results indicate that our precision medicine pipeline may be used to identify compounds capable of synergizing with inhibitors of KRAS G12C, and to assess their likelihood of becoming drugs by understanding their behavior at the proteomic/interactomic scales.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteômica , Mutação , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Combinação de MedicamentosRESUMO
BACKGROUND: T-cell longevity is undermined by antigen-driven differentiation programs that render cells prone to attrition through several mechanisms. CD8 + T cells that express the Tcf-1 transcription factor have undergone limited differentiation and exhibit stem-cell-like replenishment functions that facilitate persistence. We engineered human CD8 + T cells to constitutively express Tcf-1 and a TCR specific for the NY-ESO-1 cancer-associated antigen. Co-engineered cells were assessed for their potential for adoptive cellular immunotherapy. METHODS: Tcf-1 mRNA encoding TCF-1B and TCF-1E isoforms, along with GzmB expression were assessed in CD62L + CD57 -, CD62L - CD57 -, and CD62L - CD57 + CD8 + T cells derived from normal donor lymphocytes. The impact of stable Tcf-1B expression on CD8 + T-cell phenotype, anti-tumor activity, and cell-cycle activity was assessed in vitro and in an in vivo tumor xenograft model. RESULTS: TCF-1B and TCF-1E were dynamically regulated during self-renewal, with progeny of recently activated naïve T cells more enriched for TCF-1B mRNA. Constitutive TCF-1B expression improved the survival of TCR-engineered CD8 + T cells upon engagement with tumor cells. Tcf-1B prohibited the acquisition of a GzmB High state, and protected T cells from apoptosis associated with elicitation of effector function, and promoted stem cell-like characteristics. CONCLUSIONS: Tcf-1 protects TCR-engineered CD8 + T cells from activation induced cell death by restricting GzmB expression. Our study presents constitutive Tcf-1B expression as a potential means to impart therapeutic T cells with attributes of persistence for durable anti-tumor activity.
Assuntos
Linfócitos T CD8-Positivos , Neoplasias , Fator 1 de Transcrição de Linfócitos T , Humanos , Antígenos de Neoplasias , Granzimas/metabolismo , Receptores de Antígenos de Linfócitos T , RNA Mensageiro/metabolismo , Fator 1 de Transcrição de Linfócitos T/genética , Fator 1 de Transcrição de Linfócitos T/metabolismoRESUMO
Given prior evidence that an affected woman conveys a higher risk of ovarian cancer to her sister than to her mother, we hypothesized that there exists an X-linked variant evidenced by transmission to a woman from her paternal grandmother via her father. We ascertained 3,499 grandmother/granddaughter pairs from the Familial Ovarian Cancer Registry at the Roswell Park Cancer Institute observing 892 informative pairs with 157 affected granddaughters. We performed germline X-chromosome exome sequencing on 186 women with ovarian cancer from the registry. The rate of cancers was 28.4% in paternal grandmother/granddaughter pairs and 13.9% in maternal pairs consistent with an X-linked dominant model (Chi-square test X2 = 0.02, p = 0.89) and inconsistent with an autosomal dominant model (X2 = 20.4, p<0.001). Paternal grandmother cases had an earlier age-of-onset versus maternal cases (hazard ratio HR = 1.59, 95%CI: 1.12-2.25) independent of BRCA1/2 status. Reinforcing the X-linked hypothesis, we observed an association between prostate cancer in men and ovarian cancer in his mother and daughters (odds ratio, OR = 2.34, p = 0.034). Unaffected mothers with affected daughters produced significantly more daughters than sons (ratio = 1.96, p<0.005). We performed exome sequencing in reported BRCA negative cases from the registry. Considering age-of-onset, one missense variant (rs176026 in MAGEC3) reached chromosome-wide significance (Hazard ratio HR = 2.85, 95%CI: 1.75-4.65) advancing the age of onset by 6.7 years. In addition to the well-known contribution of BRCA, we demonstrate that a genetic locus on the X-chromosome contributes to ovarian cancer risk. An X-linked pattern of inheritance has implications for genetic risk stratification. Women with an affected paternal grandmother and sisters of affected women are at increased risk for ovarian cancer. Further work is required to validate this variant and to characterize carrier families.
Assuntos
Genes Ligados ao Cromossomo X , Hereditariedade , Neoplasias Ovarianas/epidemiologia , Neoplasias Ovarianas/genética , Herança Paterna/genética , Adulto , Idade de Início , Neoplasias da Mama/complicações , Neoplasias da Mama/epidemiologia , Neoplasias da Mama/genética , Família , Feminino , Predisposição Genética para Doença , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Genéticos , Neoplasias Ovarianas/complicações , Linhagem , Sistema de RegistrosRESUMO
Immunotherapy initially demonstrated promising results in prostate cancer (PCa), but the modest or negative results of many recent trials highlight the need to overcome the poor immunogenicity of this cancer. The design of effective therapies for PCa is challenged by the limited understanding of the interface between PCa cells and the immune system in mediating therapeutic resistance. Prompted by our recent observations that elevated WHSC1, a histone methyltransferase known to promote progression of numerous cancers, can silence antigen processing and presentation in PCa, we performed a single-cell analysis of the intratumoral immune dynamics following in vivo pharmacological inhibition of WHSC1 in mice grafted with TRAMP C2 cells. We observed an increase in cytotoxic T and NK cells accumulation and effector function, accompanied by a parallel remodeling of the myeloid compartment, as well as abundant shifts in key ligand-receptor signaling pathways highlighting changes in cell-to-cell communication driven by WHSC1 inhibition. This comprehensive profiling of both immune and molecular changes during the course of WHSC1 blockade deepens our fundamental understanding of how anti-tumor immune responses develop and can be enhanced therapeutically for PCa.
Assuntos
Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Imunoterapia , Neoplasias da Próstata/imunologia , Microambiente Tumoral , Animais , Linhagem Celular Tumoral , Masculino , Camundongos , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/fisiopatologia , Neoplasias da Próstata/terapiaRESUMO
Despite the identification of several ovarian cancer (OC) predisposition genes, a large proportion of familial OC risk remains unexplained. We adopted a two-stage design to identify new OC predisposition genes. We first carried out a large germline whole-exome sequencing study on 158 patients from 140 families with significant OC history, but without evidence of genetic predisposition due to BRCA1/2. We then evaluated the potential candidate genes in a large case-control association study involving 381 OC cases in the Cancer Genome Atlas project and 27,173 population controls from the Exome Aggregation Consortium. Two new putative OC risk genes were identified, namely, ANKRD11, a putative tumor suppressor, and POLE, an enzyme involved in DNA repair and replication. These two genes likely confer moderate OC risk. We performed in vitro experiments and showed an ANKRD11 mutation identified in our patients markedly lowered the protein expression by compromising protein stability. Upon future validation and functional characterization, these genes may shed light on cancer etiology along with improving ascertainment power and preventive care of individuals at high risk of OC.
Assuntos
Carcinoma Epitelial do Ovário/genética , Neoplasias Ovarianas/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Proteína BRCA1/genética , Proteína BRCA2/genética , Carcinoma Epitelial do Ovário/patologia , Estudos de Casos e Controles , Criança , Feminino , Genes BRCA1 , Genes BRCA2 , Predisposição Genética para Doença , Células HEK293 , Humanos , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Neoplasias Ovarianas/patologia , Proteínas Repressoras/genética , Sequenciamento do Exoma , Adulto JovemRESUMO
Exosomes, including human melanoma-derived exosomes (HMEX), are known to suppress the function of immune effector cells, which for HMEX has been associated with the surface presence of the immune checkpoint ligand PD-L1. This study investigated the relationship between the BRAF mutational status of melanoma cells and the inhibition of secreted HMEX exosomes on antigen-specific human T cells. Exosomes were isolated from two melanoma cell lines, 2183-Her4 and 888-mel, which are genetically wild-type BRAFWT and BRAFV600E, respectively. HMEX were isolated using a modified, size-exclusion chromatography (SEC) method shown to reduce co-isolation of non-exosome-associated cytokines compared to ultracentrifugation isolation. The immunoinhibitory effect of the exosomes was tested in vitro on patient-derived NY-ESO-1-specific CD8+ T cells challenged with NY-ESO-1 antigen. HMEX from both cell lines inhibited the immune response of antigen-specific T cells comparably, as evidenced by the reduction of IFN-γ and TNF-α in NY-ESO-1 tetramer-positive cells. This inhibition could be partially reversed by the presence of anti-PD-L1 and anti-IL-10 antibodies. IL-10 has been demonstrated to be a critical pathway for sustaining enhanced tumorigenesis in BRAFV600E mutant cells compared to BRAFWT melanoma cells. Thus, we demonstrate that HMEX inhibit antigen-specific T cell responses independent of the BRAF mutational status of the parent cells. In addition, PD-L1 and IL-10 contribute to the HMEX-mediated immunosuppression of antigen-specific human T cells. The inhibitory capacity of exosomes should be taken into consideration when developing therapies that are reliant upon the potency of customized, antigen-specific effector T cells.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Exossomos/metabolismo , Imunomodulação/genética , Interleucina-10/metabolismo , Mutação , Proteínas Proto-Oncogênicas B-raf/genética , Alelos , Substituição de Aminoácidos , Apoptose , Biomarcadores Tumorais , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Citometria de Fluxo , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Proteínas de Checkpoint Imunológico/metabolismo , Imunomodulação/efeitos dos fármacos , Interleucina-10/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismoRESUMO
Multiple candidate gene-association studies of non-HLA single-nucleotide polymorphisms (SNPs) and outcomes after blood or marrow transplant (BMT) have been conducted. We identified 70 publications reporting 45 SNPs in 36 genes significantly associated with disease-related mortality, progression-free survival, transplant-related mortality, and/or overall survival after BMT. Replication and validation of these SNP associations were performed using DISCOVeRY-BMT (Determining the Influence of Susceptibility COnveying Variants Related to one-Year mortality after BMT), a well-powered genome-wide association study consisting of 2 cohorts, totaling 2888 BMT recipients with acute myeloid leukemia, acute lymphoblastic leukemia, or myelodysplastic syndrome, and their HLA-matched unrelated donors, reported to the Center for International Blood and Marrow Transplant Research. Gene-based tests were used to assess the aggregate effect of SNPs on outcome. None of the previously reported significant SNPs replicated at P < .05 in DISCOVeRY-BMT. Validation analyses showed association with one previously reported donor SNP at P < .05 and survival; more associations would be anticipated by chance alone. No gene-based tests were significant at P < .05. Functional annotation with publicly available data shows these candidate SNPs most likely do not have biochemical function; only 13% of candidate SNPs correlate with gene expression or are predicted to impact transcription factor binding. Of these, half do not impact the candidate gene of interest; the other half correlate with expression of multiple genes. These findings emphasize the peril of pursing candidate approaches and the importance of adequately powered tests of unbiased genome-wide associations with BMT clinical outcomes given the ultimate goal of improving patient outcomes.
Assuntos
Transplante de Medula Óssea/mortalidade , Intervalo Livre de Doença , Estudo de Associação Genômica Ampla , Polimorfismo de Nucleotídeo Único , Transplante de Células-Tronco/mortalidade , Estudos de Validação como Assunto , Aloenxertos , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/mortalidade , Leucemia Mieloide Aguda/terapia , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/mortalidade , Síndromes Mielodisplásicas/terapia , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/mortalidade , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapiaRESUMO
Major progress in the analysis of human immune responses to cancer has been made through the molecular characterization of human tumour antigens. The development of therapeutic strategies for eliciting immune-mediated rejection of tumours has accelerated due to the elucidation of the molecular basis for tumour cell recognition and destruction by immune cells. Of the various human tumour antigens defined to date in ovarian cancer, the cancer-testis (CT) family of antigens have been studied extensively preclinically and clinically because of their testis-restricted expression in normal tissues and ability to elicit robust immune responses. Recent developments in cancer sequencing technologies offer a unique opportunity to identify tumour mutations with the highest likelihood of being expressed and recognized by the immune system. Such mutations, or neoantigens, could potentially serve as specific immune targets for T-cell-mediated destruction of cancer cells. This review will highlight current work in selecting tumour rejection antigens in ovarian cancer for improving the efficacy of immunotherapy.
Assuntos
Antígenos de Neoplasias/imunologia , Neoplasias Ovarianas/etiologia , Neoplasias Ovarianas/metabolismo , Evasão Tumoral/imunologia , Animais , Antígenos de Neoplasias/genética , Ensaios Clínicos como Assunto , Feminino , Humanos , Imunomodulação , Imunoterapia , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/terapia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Evasão Tumoral/genéticaRESUMO
To define the functions of NCOR1 we developed an integrative analysis that combined ENCODE and NCI-60 data, followed by in vitro validation. NCOR1 and H3K9me3 ChIP-Seq, FAIRE-seq and DNA CpG methylation interactions were related to gene expression using bootstrapping approaches. Most NCOR1 combinations (24/44) were associated with significantly elevated level expression of protein coding genes and only very few combinations related to gene repression. DAVID's biological process annotation revealed that elevated gene expression was uniquely associated with acetylation and ETS binding. A matrix of gene and drug interactions built on NCI-60 data identified that Imatinib significantly targeted the NCOR1 governed transcriptome. Stable knockdown of NCOR1 in K562 cells slowed growth and significantly repressed genes associated with NCOR1 cistrome, again, with the GO terms acetylation and ETS binding, and significantly dampened sensitivity to Imatinib-induced erythroid differentiation. Mining public microarray data revealed that NCOR1-targeted genes were significantly enriched in Imatinib response gene signatures in cell lines and chronic myelogenous leukemia (CML) patients. These approaches integrated cistrome, transcriptome and drug sensitivity relationships to reveal that NCOR1 function is surprisingly most associated with elevated gene expression, and that these targets, both in CML cell lines and patients, associate with sensitivity to Imatinib.
Assuntos
Células Eritroides/metabolismo , Regulação da Expressão Gênica , Mesilato de Imatinib/farmacologia , Correpressor 1 de Receptor Nuclear/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Sítios de Ligação , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular Tumoral , Epigênese Genética , Células Eritroides/efeitos dos fármacos , Genômica , Humanos , Células K562 , Correpressor 1 de Receptor Nuclear/antagonistas & inibidores , Fatores de Transcrição/metabolismoRESUMO
The current study aimed to assess the topology of the nuclear receptor (NR) superfamily in normal prostate epithelial cells and its distortion in prostate cancer. Both in vitro and in silico approaches were utilized to profile NRs expressed in non-malignant RWPE-1 cells, which were subsequently investigated by treating cells with 132 binary NR ligand combinations. Nine significant cooperative interactions emerged including both superadditive [22(R)-hydroxycholesterol and eicosatetraenoic acid] and subadditive [1α,25(OH)2D3 and chenodeoxycholic acid] cellular responses, which could be explained in part by cooperative control of cell-cycle progression and candidate gene expression. In addition, publicly available data were employed to assess NR expression in human prostate tissue. Common and significant loss of NR superfamily expression was established in publicly available data from prostate tumors, in part predicting parallel distortion of targeting microRNA. These findings suggest that the NR superfamily in the prostate cooperatively integrates signals from dietary, hormonal and metabolic cues, and is significantly distorted in prostate cancer.
Assuntos
Proliferação de Células , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Próstata/metabolismo , Neoplasias da Próstata/patologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Apoptose , Western Blotting , Células Cultivadas , Humanos , Masculino , Neoplasias da Próstata/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores Citoplasmáticos e Nucleares/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The current study investigated transcriptional distortion in prostate cancer cells using the vitamin D receptor (VDR) as a tool to examine how epigenetic events driven by corepressor binding and CpG methylation lead to aberrant gene expression. These relationships were investigated in the non-malignant RWPE-1 cells that were 1α,25(OH)(2)D(3) responsive (RWPE-1) and malignant cell lines that were 1α,25(OH)(2)D(3) partially responsive (RWPE-2) and resistant (PC-3). These studies revealed that selective attenuation and repression of VDR transcriptional responses in the cancer cell lines reflected their loss of antiproliferative sensitivity. This was evident in VDR target genes including VDR, CDKN1A (encodes p21( (waf1/cip1) )) and GADD45A; NCOR1 knockdown alleviated this malignant transrepression. ChIP assays in RWPE-1 and PC-3 cells revealed that transrepression of CDKN1A was associated with increased NCOR1 enrichment in response to 1α,25(OH)(2)D(3) treatment. These findings supported the concept that retained and increased NCOR1 binding, associated with loss of H3K9ac and increased H3K9me2, may act as a beacon for the initiation and recruitment of DNA methylation. Overexpressed histone methyltransferases (KMTs) were detectable in a wide panel of prostate cancer cell lines compared with RWPE-1 and suggested that generation of H3K9me2 states would be favored. Cotreatment of cells with the KMT inhibitor, chaetocin, increased 1α,25(OH)(2)D(3)-mediated induction of CDKN1A expression supporting a role for this event to disrupt CDKN1A regulation. Parallel surveys in PC-3 cells of CpG methylation around the VDR binding regions on CDKN1A revealed altered basal and VDR-regulated DNA methylation patterns that overlapped with VDR-induced recruitment of NCOR1 and gene transrepression. Taken together, these findings suggest that sustained corepressor interactions with nuclear-resident transcription factors may inappropriately transform transient-repressive histone states into more stable and repressive DNA methylation events.
Assuntos
Inibidor de Quinase Dependente de Ciclina p21/genética , Metilação de DNA , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Correpressor 1 de Receptor Nuclear/metabolismo , Neoplasias da Próstata/genética , Receptores de Calcitriol/metabolismo , Apoptose/efeitos dos fármacos , Conservadores da Densidade Óssea/farmacologia , Calcitriol/farmacologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Imunoprecipitação da Cromatina , Epigênese Genética , Humanos , Masculino , Correpressor 1 de Receptor Nuclear/genética , Regiões Promotoras Genéticas/genética , Próstata/metabolismo , Próstata/patologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Calcitriol/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de SinaisRESUMO
The vertebrate-restricted carcinoembryonic antigen gene family evolves extremely rapidly. Among their widely expressed members, the mammal-specific, secreted CEACAM16 is exceptionally well conserved and specifically expressed in the inner ear. To elucidate a potential auditory function, we inactivated murine Ceacam16 by homologous recombination. In young Ceacam16(-/-) mice the hearing threshold for frequencies below 10 kHz and above 22 kHz was raised. This hearing impairment progressed with age. A similar phenotype is observed in hearing-impaired members of Family 1070 with non-syndromic autosomal dominant hearing loss (DFNA4) who carry a missense mutation in CEACAM16. CEACAM16 was found in interdental and Deiters cells and was deposited in the tectorial membrane of the cochlea between postnatal days 12 and 15, when hearing starts in mice. In cochlear sections of Ceacam16(-/-) mice tectorial membranes were significantly more often stretched out as compared with wild-type mice where they were mostly contracted and detached from the outer hair cells. Homotypic cell sorting observed after ectopic cell surface expression of the carboxyl-terminal immunoglobulin variable-like N2 domain of CEACAM16 indicated that CEACAM16 can interact in trans. Furthermore, Western blot analyses of CEACAM16 under reducing and non-reducing conditions demonstrated oligomerization via unpaired cysteines. Taken together, CEACAM16 can probably form higher order structures with other tectorial membrane proteins such as α-tectorin and ß-tectorin and influences the physical properties of the tectorial membrane. Evolution of CEACAM16 might have been an important step for the specialization of the mammalian cochlea, allowing hearing over an extended frequency range.
Assuntos
Moléculas de Adesão Celular/biossíntese , Moléculas de Adesão Celular/genética , Perda Auditiva/genética , Perda Auditiva/metabolismo , Animais , Cóclea/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Feminino , Proteínas Ligadas por GPI/metabolismo , Células Ciliadas Auditivas/metabolismo , Audição , Humanos , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Mutação , Cadeias Pesadas de Miosina/genética , Miosina Tipo II/genética , Recombinação Genética , Membrana Tectorial/metabolismoRESUMO
In non-malignant RWPE-1 prostate epithelial cells signaling by the nuclear receptor Vitamin D Receptor (VDR, NR1I1) induces cell cycle arrest through targets including CDKN1A (encodes p21((waf1/cip1))). VDR dynamically induced individual histone modification patterns at three VDR binding sites (R1, 2, 3) on the CDKN1A promoter. The magnitude of these modifications was specific to each phase of the cell cycle. For example, H3K9ac enrichment occurred rapidly only at R2, whereas parallel accumulation of H3K27me3 occurred at R1; these events were significantly enriched in G(1) and S phase cells, respectively. The epigenetic events appeared to allow VDR actions to combine with p53 to enhance p21((waf1/cip1)) activation further. In parallel, VDR binding to the MCM7 gene induced H3K9ac enrichment associated with rapid mRNA up-regulation to generate miR-106b and consequently regulate p21((waf1/cip1)) expression. We conclude that VDR binding site- and promoter-specific patterns of histone modifications combine with miRNA co-regulation to form a VDR-regulated feed-forward loop to control p21((waf1/cip1)) expression and cell cycle arrest. Dissection of this feed-forward loop in a non-malignant prostate cell system illuminates mechanisms of sensitivity and therefore possible resistance in prostate and other VDR responsive cancers.
Assuntos
Inibidor de Quinase Dependente de Ciclina p21/genética , Epigênese Genética , Próstata/metabolismo , Receptores de Calcitriol/metabolismo , Animais , Calcitriol/farmacologia , Ciclo Celular/efeitos dos fármacos , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Retroalimentação Fisiológica , Histonas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/metabolismo , Regiões Promotoras Genéticas , Próstata/efeitos dos fármacos , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismoRESUMO
Tumor antigen-driven responses to weakly immunogenic self-antigens and neoantigens directly affect treatment efficacy following immunotherapy. Using orthotopically grown SV40 T antigen+ ovarian carcinoma in antigen-naive wild-type or TgMISIIR-TAg-Low transgenic mice expressing SV40 T antigen as a self-antigen, we investigated the impact of CXCR4-antagonist-armed oncolytic virotherapy on tumor progression and antitumor immunity. Immunostaining and single-cell RNA sequencing analyses of the peritoneal tumor microenvironment of untreated tumors in syngeneic wild-type mice revealed the presence of SV40 T antigen-specific CD8+ T cells, a balanced M1/M2 transcriptomic signature of tumor-associated macrophages, and immunostimulatory cancer-associated fibroblasts. This contrasted with polarized M2 tumor-associated macrophages, immunosuppressive cancer-associated fibroblasts, and poor immune activation in TgMISIIR-TAg-Low mice. Intraperitoneal delivery of CXCR4-antagonist-armed oncolytic vaccinia virus led to nearly complete depletion of cancer-associated fibroblasts, M1 polarization of macrophages, and generation of SV40 T antigen-specific CD8+ T cells in transgenic mice. Cell depletion studies revealed that the therapeutic effect of armed oncolytic virotherapy was dependent primarily on CD8+ cells. These results demonstrate that targeting the interaction between immunosuppressive cancer-associated fibroblasts and macrophages in the tolerogenic tumor microenvironment by CXCR4-A-armed oncolytic virotherapy induces tumor/self-specific CD8+ T cell responses and consequently increases therapeutic efficacy in an immunocompetent ovarian cancer model.
RESUMO
Clonotypic αß T cell responses to cargoes presented by major histocompatibility complex (MHC), MR1, or CD1 proteins underpin adaptive immunity. Those responses are mostly mediated by complementarity-determining region 3 motifs created by quasi-random T cell receptor (TCR) gene rearrangements, with diversity being highest for TCRγδ. Nonetheless, TCRγδ also displays nonclonotypic innate responsiveness following engagement of germline-encoded Vγ-specific residues by butyrophilin (BTN) or BTN-like (BTNL) proteins that uniquely mediate γδ T cell subset selection. We now report that nonclonotypic TCR engagement likewise induces distinct phenotypes in TCRαß+ cells. Specifically, antibodies to germline-encoded human TCRVß motifs consistently activated naïve or memory T cells toward core states distinct from those induced by anti-CD3 or superantigens and from others commonly reported. Those states combined selective proliferation and effector function with activation-induced inhibitory receptors and memory differentiation. Thus, nonclonotypic TCRVß targeting broadens our perspectives on human T cell response modes and might offer ways to induce clinically beneficial phenotypes in defined T cell subsets.
Assuntos
Receptores de Antígenos de Linfócitos T alfa-beta , Receptores de Antígenos de Linfócitos T gama-delta , Humanos , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Subpopulações de Linfócitos T , Butirofilinas/genética , Butirofilinas/metabolismo , Fenótipo , ImunoterapiaRESUMO
To uncover underlying mechanisms associated with failure of indoleamine 2,3-dioxygenase 1 (IDO1) blockade in clinical trials, we conducted a pilot, window-of-opportunity clinical study in 17 patients with newly diagnosed advanced high-grade serous ovarian cancer before their standard tumor debulking surgery. Patients were treated with the IDO1 inhibitor epacadostat, and immunologic, transcriptomic, and metabolomic characterization of the tumor microenvironment was undertaken in baseline and posttreatment tumor biopsies. IDO1 inhibition resulted in efficient blockade of the kynurenine pathway of tryptophan degradation and was accompanied by a metabolic adaptation that shunted tryptophan catabolism toward the serotonin pathway. This resulted in elevated nicotinamide adenine dinucleotide (NAD+), which reduced T cell proliferation and function. Because NAD+ metabolites could be ligands for purinergic receptors, we investigated the impact of blocking purinergic receptors in the presence or absence of NAD+ on T cell proliferation and function in our mouse model. We demonstrated that A2a and A2b purinergic receptor antagonists, SCH58261 or PSB1115, respectively, rescued NAD+-mediated suppression of T cell proliferation and function. Combining IDO1 inhibition and A2a/A2b receptor blockade improved survival and boosted the antitumor immune signature in mice with IDO1 overexpressing ovarian cancer. These findings elucidate the downstream adaptive metabolic consequences of IDO1 blockade in ovarian cancers that may undermine antitumor T cell responses in the tumor microenvironment.
Assuntos
Indolamina-Pirrol 2,3,-Dioxigenase , Neoplasias Ovarianas , Animais , Feminino , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Ativação Linfocitária , Camundongos , NAD , Neoplasias Ovarianas/tratamento farmacológico , Triptofano/metabolismo , Microambiente TumoralRESUMO
The immunoregulatory enzyme, indoleamine 2,3-dioxygenase (IDO1) and the PD-1/PD-L1 axis are potent mechanisms that impede effective anti-tumor immunity in ovarian cancer. However, whether the IDO pathway regulates PD-1 expression in T cells is currently unknown. Here we show that tumoral IDO1 expression led to profound changes in tryptophan, nicotinate/nicotinamide, and purine metabolic pathways in the ovarian tumor microenvironment, and to an increased frequency of PD-1+CD8+ tumor infiltrating T cells. We determined that activation of the aryl hydrocarbon receptor (AHR) by kynurenine induced PD-1 expression, and this effect was significantly abrogated by the AHR antagonist CH223191. Mechanistically, kynurenine alters chromatin accessibility in regulatory regions of T cell inhibitory receptors, allowing AHR to bind to consensus XRE motifs in the promoter region of PD-1. These results enable the design of strategies to target the IDO1 and AHR pathways for enhancing anti-tumor immunity in ovarian cancer.
Assuntos
Indolamina-Pirrol 2,3,-Dioxigenase/genética , Neoplasias Ovarianas/etiologia , Neoplasias Ovarianas/metabolismo , Receptor de Morte Celular Programada 1/genética , Receptores de Hidrocarboneto Arílico/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Animais , Sítios de Ligação , Biomarcadores , Modelos Animais de Doenças , Suscetibilidade a Doenças , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Masculino , Camundongos , Neoplasias Ovarianas/patologia , Receptor de Morte Celular Programada 1/química , Receptor de Morte Celular Programada 1/metabolismo , Ligação Proteica , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismoRESUMO
PURPOSE: Resident memory CD8 T cells, owing to their ability to reside and persist in peripheral tissues, impart adaptive sentinel activity and amplify local immune response, and have beneficial implications for tumor surveillance and control. The current study aimed to clarify the less known chemotactic mechanisms that govern the localization, retention, and residency of memory CD8 T cells in the ovarian tumor microenvironment. EXPERIMENTAL DESIGN: RNA and protein expressions of chemokine receptors in CD8+ resident memory T cells in human ovarian tumor-infiltrating CD8+ T cells and their association with survival were analyzed. The role of CXCR6 on antitumor T cells was investigated using prophylactic vaccine models in murine ovarian cancer. RESULTS: Chemokine receptor profiling of CD8+CD103+ resident memory tumor-infiltrating lymphocytes in patients with ovarian cancer revealed high expression of CXCR6. Analysis of The Cancer Genome Atlas (TCGA) (ovarian cancer database revealed CXCR6 to be associated with CD103 and increased patient survival. Functional studies in mouse models of ovarian cancer revealed that CXCR6 is a marker of resident, but not circulatory, tumor-specific memory CD8+ T cells. CXCR6-deficient tumor-specific CD8+ T cells showed reduced retention in tumor tissues, leading to diminished resident memory responses and poor control of ovarian cancer. CONCLUSIONS: CXCR6, by promoting retention in tumor tissues, serves a critical role in resident memory T cell-mediated immunosurveillance and control of ovarian cancer. Future studies warrant exploiting CXCR6 to promote resident memory responses in cancers.
Assuntos
Linfócitos T CD8-Positivos/metabolismo , Monitorização Imunológica/métodos , Neoplasias Ovarianas/genética , Receptores CXCR6/metabolismo , Animais , Feminino , Humanos , Camundongos , Camundongos Knockout , Neoplasias Ovarianas/patologia , Microambiente TumoralRESUMO
BACKGROUND: Immunotherapy in prostate cancer (PCa) lags behind the progresses obtained in other cancer types partially because of its limited immune infiltration. Tumor-resident immune cells have been detected in the prostate, but the regulatory mechanisms that govern tumor infiltration are still poorly understood. To address this gap, we investigated the role of Wolf-Hirschhorn syndrome candidate 1 (WHSC1), a histone methyltransferase enzyme that targets dimethyl and trimethyl H3K36. WHSC1 is known to promote malignant growth and progression in multiple tumors, but its role in the interface between PCa and immune system is unknown. METHODS: RNA Sequencing (RNASeq) data from patients with PCa from The Cancer Genome Atlas (TCGA) were collected and divided into top/bottom 30% based on the expression of WHSC1 and disease-free survival was calculated. Publicly available chromatin immunoprecipitation (ChIPSeq) data were obtained from Cistrome and integrated with the available RNASeq data. RNASeq, ATACSeq and methylomic were analyzed using R Bioconductor packages comparing C42 cells with or without stable knockdown on WHSC1. Flow cytometry was used to measure Major Histocompatibility complex (MHC) levels, MHC-bound ovalbumin and tumor infiltration. C57B6 and NOD scid gamma (NSG) mice were subcutaneously grafted with TRansgenic Adenocarcinoma of the Mouse Prostate (TRAMP) C2 cells and treated with MCTP39 (10 mg/kg); tumor size was monitored over time and curves were compared using permutation analyses. All analyses used a significance threshold of 0.05. RESULTS: Leveraging TCGA data, we demonstrated that elevated WHSC1 levels positively correlate with the presence of an immunosuppressive microenvironment. We validated those results in vitro, demonstrating that genetic and pharmacological inhibition of WHSC1 restores antigen presentation. This occurs via an elegant epigenetic regulation of gene expression at the chromatin and DNA methylation levels. In vivo studies in immunocompetent mice also show an increased frequency of CD8+ T cells in tumors from mice treated with WHSC1 inhibitor, supporting the hypothesis that the antitumor effect following WHSC1 inhibition requires a fully functional immune system. CONCLUSIONS: This study demonstrates a novel role for WHSC1 in defining immune infiltration in PCa, with significant future implications for the use of immunotherapies in prostate malignancies.