RESUMO
Early in the COVID-19 pandemic, data suggested that males had a higher risk of developing severe disease and that androgen deprivation therapy might be associated with protection. Combined with the fact that TMPRSS2 (transmembrane serine protease 2), a host entry factor for the SARS-CoV-2 virus, was a well-known androgen-regulated gene, this led to an upsurge of research investigating androgen receptor (AR)-targeting drugs. Proxalutamide, an AR antagonist, was shown in initial clinical studies to benefit COVID-19 patients; however, further validation is needed as one study was retracted. Due to continued interest in proxalutamide, which is in phase 3 trials, we examined its ability to impact SARS-CoV-2 infection and downstream inflammatory responses. Proxalutamide exerted similar effects as enzalutamide, an AR antagonist prescribed for advanced prostate cancer, in decreasing AR signaling and expression of TMPRSS2 and angiotensin-converting enzyme 2 (ACE2), the SARS-CoV-2 receptor. However, proxalutamide led to degradation of AR protein, which was not observed with enzalutamide. Proxalutamide inhibited SARS-CoV-2 infection with an IC50 value of 97 nM, compared to 281 nM for enzalutamide. Importantly, proxalutamide inhibited infection by multiple SARS-CoV-2 variants and synergized with remdesivir. Proxalutamide protected against cell death in response to tumor necrosis factor alpha and interferon gamma, and overall survival of mice was increased with proxalutamide treatment prior to cytokine exposure. Mechanistically, we found that proxalutamide increased levels of NRF2, an essential transcription factor that mediates antioxidant responses, and decreased lung inflammation. These data provide compelling evidence that proxalutamide can prevent SARS-CoV-2 infection and cytokine-induced lung damage, suggesting that promising clinical data may emerge from ongoing phase 3 trials.
Assuntos
COVID-19 , Neoplasias da Próstata , Masculino , Humanos , Animais , Camundongos , SARS-CoV-2/metabolismo , Androgênios , Antagonistas de Androgênios/uso terapêutico , Pandemias , Peptidil Dipeptidase A/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Interferon gama/uso terapêuticoRESUMO
Although the COVID-19 pandemic began over three years ago, the virus responsible for the disease, SARS-CoV-2, continues to infect people across the globe. As such, there remains a critical need for development of novel therapeutics against SARS-CoV-2. One technology that has remained relatively unexplored in COVID-19 is the use of antisense oligonucleotides (ASOs)-short single-stranded nucleic acids that bind to target RNA transcripts to modulate their expression. In this study, ASOs targeted against the SARS-CoV-2 genome and host entry factors, ACE2 and TMPRSS2, were designed and tested for their ability to inhibit cellular infection by SARS-CoV-2. Using our previously developed SARS-CoV-2 bioassay platform, we screened 180 total ASOs targeting various regions of the SARS-CoV-2 genome and validated several ASOs that potently blocked SARS-CoV-2 infection in vitro. Notably, select ASOs retained activity against both the WA1 and B.1.1.7 (commonly known as alpha) variants. Screening of ACE2 and TMPRSS2 ASOs showed that targeting of ACE2 also potently prevented infection by the WA1 and B.1.1.7 SARS-CoV-2 viruses in the tested cell lines. Combined with the demonstrated success of ASOs in other disease indications, these results support further research into the development of ASOs targeting SARS-CoV-2 and host entry factors as potential COVID-19 therapeutics.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , Enzima de Conversão de Angiotensina 2/metabolismo , Oligonucleotídeos Antissenso/farmacologia , Oligonucleotídeos Antissenso/uso terapêutico , Pandemias , Peptidil Dipeptidase A/metabolismo , Internalização do VírusRESUMO
Multi-tyrosine kinase inhibitors (MTKIs) have thus far had limited success in the treatment of castration-resistant prostate cancer (CRPC). Here, we report a phase I-cleared orally bioavailable MTKI, ESK981, with a novel autophagy inhibitory property that decreased tumor growth in diverse preclinical models of CRPC. The anti-tumor activity of ESK981 was maximized in immunocompetent tumor environments where it upregulated CXCL10 expression through the interferon gamma pathway and promoted functional T cell infiltration, which resulted in enhanced therapeutic response to immune checkpoint blockade. Mechanistically, we identify the lipid kinase PIKfyve as the direct target of ESK981. PIKfyve-knockdown recapitulated ESK981's anti-tumor activity and enhanced the therapeutic benefit of immune checkpoint blockade. Our study reveals that targeting PIKfyve via ESK981 turns tumors from cold into hot through inhibition of autophagy, which may prime the tumor immune microenvironment in advanced prostate cancer patients and be an effective treatment strategy alone or in combination with immunotherapies.
Assuntos
Inibidores de Checkpoint Imunológico , Neoplasias de Próstata Resistentes à Castração , Autofagia , Humanos , Imunoterapia/métodos , Masculino , Fosfatidilinositol 3-Quinases/farmacologia , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Microambiente TumoralRESUMO
Diverse subtypes of renal cell carcinomas (RCCs) display a wide spectrum of histomorphologies, proteogenomic alterations, immune cell infiltration patterns, and clinical behavior. Delineating the cells of origin for different RCC subtypes will provide mechanistic insights into their diverse pathobiology. Here, we employed single-cell RNA sequencing (scRNA-seq) to develop benign and malignant renal cell atlases. Using a random forest model trained on this cell atlas, we predicted the putative cell of origin for more than 10 RCC subtypes. scRNA-seq also revealed several attributes of the tumor microenvironment in the most common subtype of kidney cancer, clear cell RCC (ccRCC). We elucidated an active role for tumor epithelia in promoting immune cell infiltration, potentially explaining why ccRCC responds to immune checkpoint inhibitors, despite having a low neoantigen burden. In addition, we characterized an association between high endothelial cell types and lack of response to immunotherapy in ccRCC. Taken together, these single-cell analyses of benign kidney and RCC provide insight into the putative cell of origin for RCC subtypes and highlight the important role of the tumor microenvironment in influencing ccRCC biology and response to therapy.
Assuntos
Carcinoma de Células Renais/patologia , Carcinoma de Células Renais/terapia , Neoplasias Renais/patologia , Neoplasias Renais/terapia , Análise de Célula Única , Carcinoma de Células Renais/imunologia , Sobrevivência Celular , Células Endoteliais/patologia , Células Epiteliais/patologia , Humanos , Imunoterapia , Rim/patologia , Neoplasias Renais/imunologia , Linfócitos do Interstício Tumoral/imunologia , Células Mieloides/patologia , Resultado do TratamentoRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for COVID-19, employs two key host proteins to gain entry and replicate within cells, angiotensin-converting enzyme 2 (ACE2) and the cell surface transmembrane protease serine 2 (TMPRSS2). TMPRSS2 was first characterized as an androgen-regulated gene in the prostate. Supporting a role for sex hormones, males relative to females are disproportionately affected by COVID-19 in terms of mortality and morbidity. Several studies, including one employing a large epidemiological cohort, suggested that blocking androgen signaling is protective against COVID-19. Here, we demonstrate that androgens regulate the expression of ACE2, TMPRSS2, and androgen receptor (AR) in subsets of lung epithelial cells. AR levels are markedly elevated in males relative to females greater than 70 y of age. In males greater than 70 y old, smoking was associated with elevated levels of AR and ACE2 in lung epithelial cells. Transcriptional repression of the AR enhanceosome with AR or bromodomain and extraterminal domain (BET) antagonists inhibited SARS-CoV-2 infection in vitro. Taken together, these studies support further investigation of transcriptional inhibition of critical host factors in the treatment or prevention of COVID-19.
RESUMO
Both KRAS and EGFR are essential mediators of pancreatic cancer development and interact with Argonaute 2 (AGO2) to perturb its function. Here, in a mouse model of mutant KRAS-driven pancreatic cancer, loss of AGO2 allows precursor lesion (PanIN) formation yet prevents progression to pancreatic ductal adenocarcinoma (PDAC). Precursor lesions with AGO2 ablation undergo oncogene-induced senescence with altered microRNA expression and EGFR/RAS signaling, bypassed by loss of p53. In mouse and human pancreatic tissues, PDAC progression is associated with increased plasma membrane localization of RAS/AGO2. Furthermore, phosphorylation of AGO2Y393 disrupts both the wild-type and oncogenic KRAS-AGO2 interaction, albeit under different conditions. ARS-1620 (G12C-specific inhibitor) disrupts the KRASG12C-AGO2 interaction, suggesting that the interaction is targetable. Altogether, our study supports a biphasic model of pancreatic cancer development: an AGO2-independent early phase of PanIN formation reliant on EGFR-RAS signaling, and an AGO2-dependent phase wherein the mutant KRAS-AGO2 interaction is critical for PDAC progression.
Assuntos
Proteínas Argonautas/metabolismo , Neoplasias Pancreáticas/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Alelos , Animais , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Senescência Celular , Progressão da Doença , Receptores ErbB/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Genótipo , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Transplante de Neoplasias , Neoplasias Pancreáticas/patologia , Fosforilação , Ligação Proteica , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismoRESUMO
The clinical utility of inhibiting cytochrome P450 17A1 (CYP17), a cytochrome p450 enzyme that is required for the production of androgens, has been exemplified by the approval of abiraterone for the treatment of castration-resistant prostate cancer (CRPC). Recently, however, it has been reported that CYP17 inhibitors can interact directly with the androgen receptor (AR). A phase I study recently reported that seviteronel, a CYP17 lyase-selective inhibitor, ædemonstrated a sustained reduction in prostate-specific antigen in a patient with CRPC, and another study showed seviteronel's direct effects on AR function. This suggested that seviteronel may have therapeutically relevant activities in addition to its ability to inhibit androgen production. Here, we have demonstrated that CYP17 inhibitors, with the exception of orteronel, can function as competitive AR antagonists. Conformational profiling revealed that the CYP17 inhibitor-bound AR adopted a conformation that resembled the unliganded AR (apo-AR), precluding nuclear localization and DNA binding. Further, we observed that seviteronel and abiraterone inhibited the growth of tumor xenografts expressing the clinically relevant mutation AR-F876L and that this activity could be attributed entirely to competitive AR antagonism. The results of this study suggest that the ability of CYP17 inhibitors to directly antagonize the AR may contribute to their clinical efficacy in CRPC.
Assuntos
Antagonistas de Receptores de Andrógenos/farmacologia , Antineoplásicos Hormonais/farmacologia , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Esteroide 17-alfa-Hidroxilase/antagonistas & inibidores , Transporte Ativo do Núcleo Celular , Animais , Benzamidas , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Células HEK293 , Humanos , Concentração Inibidora 50 , Masculino , Metribolona/farmacologia , Camundongos Endogâmicos NOD , Camundongos SCID , Nitrilas , Feniltioidantoína/análogos & derivados , Feniltioidantoína/farmacologia , Ligação Proteica , Receptores Androgênicos/metabolismo , Esteroide 17-alfa-Hidroxilase/metabolismo , Testosterona/farmacologia , Ativação Transcricional/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Clinical resistance to the second-generation antiandrogen enzalutamide in castration-resistant prostate cancer (CRPC), despite persistent androgen receptor (AR) activity in tumors, highlights an unmet medical need for next-generation antagonists. We have identified and characterized tetra-aryl cyclobutanes (CBs) as a new class of competitive AR antagonists that exhibit a unique mechanism of action. These CBs are structurally distinct from current antiandrogens (hydroxyflutamide, bicalutamide, and enzalutamide) and inhibit AR-mediated gene expression, cell proliferation, and tumor growth in several models of CRPC. Conformational profiling revealed that CBs stabilize an AR conformation resembling an unliganded receptor. Using a variety of techniques, it was determined that the AR-CB complex was not recruited to AR-regulated promoters and, like apo AR, remains sequestered in the cytoplasm, bound to heat shock proteins. Thus, we have identified third-generation AR antagonists whose unique mechanism of action suggests that they may have therapeutic potential in CRPC.
Assuntos
Antagonistas de Receptores de Andrógenos/farmacologia , Antineoplásicos/farmacologia , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/metabolismo , Receptores Androgênicos/metabolismo , Antagonistas de Receptores de Andrógenos/química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Masculino , Neoplasias de Próstata Resistentes à Castração/patologia , Relação Estrutura-AtividadeRESUMO
Animal domestication has resulted in changes in growth and size. It has been suggested that this may have involved selection for differences in appetite. Divergent growth between chickens selected for egg laying or meat production is one such example. The neurons expressing AGRP and POMC in the basal hypothalamus are important components of appetite regulation, as are the satiety feedback pathways that carry information from the intestine, including CCK and its receptor CCKAR (CCK1 receptor). Using 16 generations of a cross between a fast and a relatively slow growing strain of chicken has identified a region on chromosome 4 downstream of the CCKAR gene, which is responsible for up to a 19% difference in body weight at 12 wk of age. Animals possessing the high-growth haplotype at the locus have lower expression of mRNA and immunoreactive CCKAR in the brain, intestine, and exocrine organs, which is correlated with increased levels of orexigenic AGRP in the hypothalamus. Animals with the high-growth haplotype are resistant to the anorectic effect of exogenously administered CCK, suggesting that their satiety set point has been altered. Comparison with traditional breeds shows that the high-growth haplotype has been present in the founders of modern meat-type strains and may have been selected early in domestication. This is the first dissection of the physiological consequences of a genetic locus for a quantitative trait that alters appetite and gives us an insight into the domestication of animals. This will allow elucidation of how differences in appetite occur in birds and also mammals.
Assuntos
Animais Domésticos , Peso Corporal/genética , Peso Corporal/fisiologia , Galinhas/genética , Galinhas/fisiologia , Crescimento/genética , Crescimento/fisiologia , Receptor de Colecistocinina A/biossíntese , Receptor de Colecistocinina A/fisiologia , Resposta de Saciedade/fisiologia , Proteína Relacionada com Agouti/biossíntese , Proteína Relacionada com Agouti/genética , Alelos , Animais , Química Encefálica/fisiologia , Cruzamentos Genéticos , Ingestão de Alimentos/genética , Ingestão de Alimentos/fisiologia , Feminino , Genótipo , Imuno-Histoquímica , Masculino , Polimorfismo de Nucleotídeo Único/genética , RNA/biossíntese , RNA/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real , Receptor de Colecistocinina A/genética , Distribuição Tecidual , Transcrição GênicaRESUMO
Estrogen receptor-alpha (ERalpha) is essential in the maintenance of cellular responsiveness to estrogen in the reproductive system. It is established that ligand binding induces downregulation of ERalpha protein by targeting receptor for destruction by the 26S proteasome. However, ERalpha is preserved in cells chronically exposed to estrogen and it is unknown how receptor levels are maintained in the continued presence of the signal that induces degradation. A modified pulse-chase analysis was developed using a tet-inducible ERalpha expression system to determine the rate of ERalpha protein decay following both acute and chronic estrogen treatments. Upon initial hormone treatment, ERalpha half-life is shortened from 3 to 1 h. However, ERalpha half-life increases over time, achieving a half-life of approximately 6 h in 72 h of estrogen treatment. Analysis of ERalpha half-life in the presence and absence of proteasome inhibitor, MG132, revealed that the increased stability is due in part to a decreased rate of proteolysis. In addition, we observed a time-dependent increase in phospho-S118 ERalpha and showed that the half-life of the phosphomimetic ERalpha mutant, S118E-ER, is identical to that of wild-type receptor under conditions of chronic estrogen treatment. These data provide evidence that as cells adapt to chronic stimulation, ERalpha protein is stabilized due first to a decreased rate of proteolysis, and secondarily, to the accumulation of proteasome-resistant, phosphorylated form of receptor. This temporal control of proteolysis allows for the establishment of steady-state levels of receptor and provides a protective mechanism against loss of hormone responsiveness.