RESUMO
The role of long noncoding RNAs (lncRNAs) in disease is incompletely understood, but their regulation of inflammation is increasingly appreciated. We addressed the extent of lncRNA involvement in inflammatory bowel disease (IBD) using biopsy-derived RNA-sequencing data from a large cohort of deeply phenotyped patients with IBD. Weighted gene correlation network analysis revealed gene modules of lncRNAs coexpressed with protein-coding genes enriched for biological pathways, correlated with epithelial and immune cell signatures, or correlated with distal colon expression. Correlation of modules with clinical features uncovered a module correlated with disease severity, with an enriched interferon response signature containing the hub lncRNA IRF1-AS1. Connecting genes to IBD-associated single nucleotide polymorphisms (SNPs) revealed an enrichment of SNP-adjacent lncRNAs in biologically relevant modules. Ulcerative colitis-specific SNPs were enriched in distal colon-related modules, suggesting that disease-specific mechanisms may result from altered lncRNA expression. The function of the IBD-associated SNP-adjacent lncRNA IRF1-AS1 was explored in human myeloid cells, and our results suggested IRF1-AS1 promoted optimal production of TNF-α, IL-6, and IL-23. A CRISPR/Cas9-mediated activation screen in THP-1 cells revealed several lncRNAs that modulated LPS-induced TNF-α responses. Overall, this study uncovered the expression patterns of lncRNAs in IBD that identify functional, disease-relevant lncRNAs.
Assuntos
Colite Ulcerativa , RNA Longo não Codificante , Humanos , Redes Reguladoras de Genes , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Fator de Necrose Tumoral alfa/genética , Colite Ulcerativa/genética , InflamaçãoRESUMO
A major challenge in inflammatory bowel disease (IBD) is the integration of diverse IBD data sets to construct predictive models of IBD. We present a predictive model of the immune component of IBD that informs causal relationships among loci previously linked to IBD through genome-wide association studies (GWAS) using functional and regulatory annotations that relate to the cells, tissues, and pathophysiology of IBD. Our model consists of individual networks constructed using molecular data generated from intestinal samples isolated from three populations of patients with IBD at different stages of disease. We performed key driver analysis to identify genes predicted to modulate network regulatory states associated with IBD, prioritizing and prospectively validating 12 of the top key drivers experimentally. This validated key driver set not only introduces new regulators of processes central to IBD but also provides the integrated circuits of genetic, molecular, and clinical traits that can be directly queried to interrogate and refine the regulatory framework defining IBD.
Assuntos
Redes Reguladoras de Genes , Genes Reguladores , Genômica/métodos , Doenças Inflamatórias Intestinais/genética , Modelos Genéticos , Transferência Adotiva , Animais , Causalidade , Células Cultivadas , Colite/induzido quimicamente , Colite/genética , Conjuntos de Dados como Assunto , Modelos Animais de Doenças , Feminino , Técnicas de Silenciamento de Genes , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Humanos , Mucosa Intestinal/metabolismo , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Interferente Pequeno/genética , Subpopulações de Linfócitos T/transplante , TranscriptomaRESUMO
The acquisition of apoptosis resistance is a fundamental event in cancer development. Among the mechanisms used by cancer cells to evade apoptosis is the dysregulation of inhibitor of apoptosis (IAP) proteins. The activity of the IAPs is regulated by endogenous IAP antagonists such as SMAC (also termed DIABLO). Antagonism of IAP proteins by SMAC occurs via binding of the N-terminal tetrapeptide (AVPI) of SMAC to selected BIR domains of the IAPs. Small molecule compounds that mimic the AVPI motif of SMAC have been designed to overcome IAP-mediated apoptosis resistance of cancer cells. Here, we report the preclinical characterization of birinapant (TL32711), a bivalent SMAC-mimetic compound currently in clinical trials for the treatment of cancer. Birinapant bound to the BIR3 domains of cIAP1, cIAP2, XIAP, and the BIR domain of ML-IAP in vitro and induced the autoubiquitylation and proteasomal degradation of cIAP1 and cIAP2 in intact cells, which resulted in formation of a RIPK1:caspase-8 complex, caspase-8 activation, and induction of tumor cell death. Birinapant preferentially targeted the TRAF2-associated cIAP1 and cIAP2 with subsequent inhibition of TNF-induced NF-κB activation. The activity of a variety of chemotherapeutic cancer drugs was potentiated by birinapant both in a TNF-dependent or TNF-independent manner. Tumor growth in multiple primary patient-derived xenotransplant models was inhibited by birinapant at well-tolerated doses. These results support the therapeutic combination of birinapant with multiple chemotherapies, in particular, those therapies that can induce TNF secretion.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Dipeptídeos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Indóis/farmacologia , Animais , Neoplasias da Mama/patologia , Caspase 8/metabolismo , Linhagem Celular Tumoral , Sinergismo Farmacológico , Feminino , Humanos , Proteínas Inibidoras de Apoptose/metabolismo , Camundongos Nus , Proteínas Mitocondriais/metabolismo , Receptores do Fator de Necrose Tumoral , Transdução de Sinais/efeitos dos fármacos , Fator 2 Associado a Receptor de TNF/metabolismoRESUMO
Birinapant (1) is a second-generation bivalent antagonist of IAP proteins that is currently undergoing clinical development for the treatment of cancer. Using a range of assays that evaluated cIAP1 stability and oligomeric state, we demonstrated that 1 stabilized the cIAP1-BUCR (BIR3-UBA-CARD-RING) dimer and promoted autoubiquitylation of cIAP1 in vitro. Smac-mimetic 1-induced loss of cIAPs correlated with inhibition of TNF-mediated NF-κB activation, caspase activation, and tumor cell killing. Many first-generation Smac-mimetics such as compound A (2) were poorly tolerated. Notably, animals that lack functional cIAP1, cIAP2, and XIAP are not viable, and 2 mimicked features of triple IAP knockout cells in vitro. The improved tolerability of 1 was associated with (i) decreased potency against cIAP2 and affinity for XIAP BIR3 and (ii) decreased ability to inhibit XIAP-dependent signaling pathways. The P2' position of 1 was critical to this differential activity, and this improved tolerability has allowed 1 to proceed into clinical studies.
Assuntos
Antineoplásicos/farmacologia , Proteínas de Transporte/química , Dipeptídeos/farmacologia , Neoplasias Hematológicas/tratamento farmacológico , Indóis/farmacologia , Proteínas Mitocondriais/química , Mimetismo Molecular , Neoplasias Experimentais/tratamento farmacológico , Animais , Antineoplásicos/uso terapêutico , Proteínas Reguladoras de Apoptose , Dipeptídeos/uso terapêutico , Descoberta de Drogas , Indóis/uso terapêutico , Camundongos , Modelos MolecularesRESUMO
PURPOSE: Inhibitor of apoptosis proteins (IAP) promote cancer cell survival and confer resistance to therapy. We report on the ability of second mitochondria-derived activator of caspases mimetic, birinapant, which acts as antagonist to cIAP1 and cIAP2, to restore the sensitivity to apoptotic stimuli such as TNF-α in melanomas. EXPERIMENTAL DESIGN: Seventeen melanoma cell lines, representing five major genetic subgroups of cutaneous melanoma, were treated with birinapant as a single agent or in combination with TNF-α. Effects on cell viability, target inhibition, and initiation of apoptosis were assessed and findings were validated in 2-dimensional (2D), 3D spheroid, and in vivo xenograft models. RESULTS: When birinapant was combined with TNF-α, strong combination activity, that is, neither compound was effective individually but the combination was highly effective, was observed in 12 of 18 cell lines. This response was conserved in spheroid models, whereas in vivo birinapant inhibited tumor growth without adding TNF-α in in vitro resistant cell lines. Birinapant combined with TNF-α inhibited the growth of a melanoma cell line with acquired resistance to BRAF inhibition to the same extent as in the parental cell line. CONCLUSIONS: Birinapant in combination with TNF-α exhibits a strong antimelanoma effect in vitro. Birinapant as a single agent shows in vivo antitumor activity, even if cells are resistant to single agent therapy in vitro. Birinapant in combination with TNF-α is effective in a melanoma cell line with acquired resistance to BRAF inhibitors.