RESUMO
Cancer cells can evade natural killer (NK) cell activity, thereby limiting anti-tumor immunity. To reveal genetic determinants of susceptibility to NK cell activity, we examined interacting NK cells and blood cancer cells using single-cell and genome-scale functional genomics screens. Interaction of NK and cancer cells induced distinct activation and type I interferon (IFN) states in both cell types depending on the cancer cell lineage and molecular phenotype, ranging from more sensitive myeloid to less sensitive B-lymphoid cancers. CRISPR screens in cancer cells uncovered genes regulating sensitivity and resistance to NK cell-mediated killing, including adhesion-related glycoproteins, protein fucosylation genes, and transcriptional regulators, in addition to confirming the importance of antigen presentation and death receptor signaling pathways. CRISPR screens with a single-cell transcriptomic readout provided insight into underlying mechanisms, including regulation of IFN-γ signaling in cancer cells and NK cell activation states. Our findings highlight the diversity of mechanisms influencing NK cell susceptibility across different cancers and provide a resource for NK cell-based therapies.
Assuntos
Neoplasias Hematológicas , Neoplasias , Humanos , Células Matadoras Naturais , Neoplasias/genética , Apresentação de Antígeno , Genômica , Citotoxicidade Imunológica/genética , Linhagem Celular TumoralRESUMO
A hallmark of high-risk childhood medulloblastoma is the dysregulation of RNA translation. Currently, it is unknown whether medulloblastoma dysregulates the translation of putatively oncogenic non-canonical open reading frames (ORFs). To address this question, we performed ribosome profiling of 32 medulloblastoma tissues and cell lines and observed widespread non-canonical ORF translation. We then developed a stepwise approach using multiple CRISPR-Cas9 screens to elucidate non-canonical ORFs and putative microproteins implicated in medulloblastoma cell survival. We determined that multiple lncRNA-ORFs and upstream ORFs (uORFs) exhibited selective functionality independent of main coding sequences. A microprotein encoded by one of these ORFs, ASNSD1-uORF or ASDURF, was upregulated, associated with MYC-family oncogenes, and promoted medulloblastoma cell survival through engagement with the prefoldin-like chaperone complex. Our findings underscore the fundamental importance of non-canonical ORF translation in medulloblastoma and provide a rationale to include these ORFs in future studies seeking to define new cancer targets.
Assuntos
Neoplasias Cerebelares , Meduloblastoma , Humanos , Biossíntese de Proteínas , Meduloblastoma/genética , Fases de Leitura Aberta/genética , Sobrevivência Celular/genética , Neoplasias Cerebelares/genéticaRESUMO
RAS oncogenes (collectively NRAS, HRAS and especially KRAS) are among the most frequently mutated genes in cancer, with common driver mutations occurring at codons 12, 13 and 611. Small molecule inhibitors of the KRAS(G12C) oncoprotein have demonstrated clinical efficacy in patients with multiple cancer types and have led to regulatory approvals for the treatment of non-small cell lung cancer2,3. Nevertheless, KRASG12C mutations account for only around 15% of KRAS-mutated cancers4,5, and there are no approved KRAS inhibitors for the majority of patients with tumours containing other common KRAS mutations. Here we describe RMC-7977, a reversible, tri-complex RAS inhibitor with broad-spectrum activity for the active state of both mutant and wild-type KRAS, NRAS and HRAS variants (a RAS(ON) multi-selective inhibitor). Preclinically, RMC-7977 demonstrated potent activity against RAS-addicted tumours carrying various RAS genotypes, particularly against cancer models with KRAS codon 12 mutations (KRASG12X). Treatment with RMC-7977 led to tumour regression and was well tolerated in diverse RAS-addicted preclinical cancer models. Additionally, RMC-7977 inhibited the growth of KRASG12C cancer models that are resistant to KRAS(G12C) inhibitors owing to restoration of RAS pathway signalling. Thus, RAS(ON) multi-selective inhibitors can target multiple oncogenic and wild-type RAS isoforms and have the potential to treat a wide range of RAS-addicted cancers with high unmet clinical need. A related RAS(ON) multi-selective inhibitor, RMC-6236, is currently under clinical evaluation in patients with KRAS-mutant solid tumours (ClinicalTrials.gov identifier: NCT05379985).
Assuntos
Antineoplásicos , Mutação , Neoplasias , Proteína Oncogênica p21(ras) , Proteínas Proto-Oncogênicas p21(ras) , Animais , Humanos , Camundongos , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Guanosina Trifosfato/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , Proteína Oncogênica p21(ras)/antagonistas & inibidores , Proteína Oncogênica p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Broad-spectrum RAS inhibition has the potential to benefit roughly a quarter of human patients with cancer whose tumours are driven by RAS mutations1,2. RMC-7977 is a highly selective inhibitor of the active GTP-bound forms of KRAS, HRAS and NRAS, with affinity for both mutant and wild-type variants3. More than 90% of cases of human pancreatic ductal adenocarcinoma (PDAC) are driven by activating mutations in KRAS4. Here we assessed the therapeutic potential of RMC-7977 in a comprehensive range of PDAC models. We observed broad and pronounced anti-tumour activity across models following direct RAS inhibition at exposures that were well-tolerated in vivo. Pharmacological analyses revealed divergent responses to RMC-7977 in tumour versus normal tissues. Treated tumours exhibited waves of apoptosis along with sustained proliferative arrest, whereas normal tissues underwent only transient decreases in proliferation, with no evidence of apoptosis. In the autochthonous KPC mouse model, RMC-7977 treatment resulted in a profound extension of survival followed by on-treatment relapse. Analysis of relapsed tumours identified Myc copy number gain as a prevalent candidate resistance mechanism, which could be overcome by combinatorial TEAD inhibition in vitro. Together, these data establish a strong preclinical rationale for the use of broad-spectrum RAS-GTP inhibition in the setting of PDAC and identify a promising candidate combination therapeutic regimen to overcome monotherapy resistance.
Assuntos
Antineoplásicos , Carcinoma Ductal Pancreático , Guanosina Trifosfato , Neoplasias Pancreáticas , Proteínas Proto-Oncogênicas p21(ras) , Animais , Feminino , Humanos , Camundongos , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/patologia , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Variações do Número de Cópias de DNA , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Genes myc , Guanosina Trifosfato/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Recidiva Local de Neoplasia/tratamento farmacológico , Recidiva Local de Neoplasia/genética , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Resultado do Tratamento , Ensaios Antitumorais Modelo de Xenoenxerto , MutaçãoRESUMO
Immune checkpoint blockade is effective for some patients with cancer, but most are refractory to current immunotherapies and new approaches are needed to overcome resistance1,2. The protein tyrosine phosphatases PTPN2 and PTPN1 are central regulators of inflammation, and their genetic deletion in either tumour cells or immune cells promotes anti-tumour immunity3-6. However, phosphatases are challenging drug targets; in particular, the active site has been considered undruggable. Here we present the discovery and characterization of ABBV-CLS-484 (AC484), a first-in-class, orally bioavailable, potent PTPN2 and PTPN1 active-site inhibitor. AC484 treatment in vitro amplifies the response to interferon and promotes the activation and function of several immune cell subsets. In mouse models of cancer resistant to PD-1 blockade, AC484 monotherapy generates potent anti-tumour immunity. We show that AC484 inflames the tumour microenvironment and promotes natural killer cell and CD8+ T cell function by enhancing JAK-STAT signalling and reducing T cell dysfunction. Inhibitors of PTPN2 and PTPN1 offer a promising new strategy for cancer immunotherapy and are currently being evaluated in patients with advanced solid tumours (ClinicalTrials.gov identifier NCT04777994 ). More broadly, our study shows that small-molecule inhibitors of key intracellular immune regulators can achieve efficacy comparable to or exceeding that of antibody-based immune checkpoint blockade in preclinical models. Finally, to our knowledge, AC484 represents the first active-site phosphatase inhibitor to enter clinical evaluation for cancer immunotherapy and may pave the way for additional therapeutics that target this important class of enzymes.
Assuntos
Imunoterapia , Neoplasias , Proteína Tirosina Fosfatase não Receptora Tipo 1 , Proteína Tirosina Fosfatase não Receptora Tipo 2 , Animais , Humanos , Camundongos , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos , Inibidores de Checkpoint Imunológico , Imunoterapia/métodos , Interferons/imunologia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Neoplasias/imunologia , Proteína Tirosina Fosfatase não Receptora Tipo 1/antagonistas & inibidores , Proteína Tirosina Fosfatase não Receptora Tipo 2/antagonistas & inibidores , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologiaRESUMO
Most deaths from cancer are explained by metastasis, and yet large-scale metastasis research has been impractical owing to the complexity of in vivo models. Here we introduce an in vivo barcoding strategy that is capable of determining the metastatic potential of human cancer cell lines in mouse xenografts at scale. We validated the robustness, scalability and reproducibility of the method and applied it to 500 cell lines1,2 spanning 21 types of solid tumour. We created a first-generation metastasis map (MetMap) that reveals organ-specific patterns of metastasis, enabling these patterns to be associated with clinical and genomic features. We demonstrate the utility of MetMap by investigating the molecular basis of breast cancers capable of metastasizing to the brain-a principal cause of death in patients with this type of cancer. Breast cancers capable of metastasizing to the brain showed evidence of altered lipid metabolism. Perturbation of lipid metabolism in these cells curbed brain metastasis development, suggesting a therapeutic strategy to combat the disease and demonstrating the utility of MetMap as a resource to support metastasis research.
Assuntos
Neoplasias da Mama/patologia , Movimento Celular , Metástase Neoplásica/patologia , Especificidade de Órgãos , Animais , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/secundário , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Processamento Eletrônico de Dados , Feminino , Xenoenxertos , Humanos , Metabolismo dos Lipídeos/genética , Camundongos , Tipagem Molecular , Mutação , Metástase Neoplásica/genética , Transplante de Neoplasias , Projetos PilotoRESUMO
NADPH oxidases (NOXs) are transmembrane enzymes that are devoted to the production of reactive oxygen species (ROS). In cancers, dysregulation of NOX enzymes affects ROS production, leading to redox unbalance and tumor progression. Consequently, NOXs are a drug target for cancer therapeutics, although current therapies have off-target effects: there is a need for isoenzyme-selective inhibitors. Here, we describe fully validated human NOX inhibitors, obtained from an in silico screen, targeting the active site of Cylindrospermum stagnale NOX5 (csNOX5). The hits are validated by in vitro and in cellulo enzymatic and binding assays, and their binding modes to the dehydrogenase domain of csNOX5 studied via high-resolution crystal structures. A high-throughput screen in a panel of cancer cells shows activity in selected cancer cell lines and synergistic effects with KRAS modulators. Our work lays the foundation for the development of inhibitor-based methods for controlling the tightly regulated and highly localized ROS sources.
Assuntos
NADPH Oxidases , Neoplasias , Humanos , NADPH Oxidases/química , NADPH Oxidases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Neoplasias/tratamento farmacológico , Oxirredução , Linhagem CelularRESUMO
Aberrant DNA repair is a hallmark of cancer, and many tumors display reduced DNA repair capacities that sensitize them to genotoxins. Here, we demonstrate that the differential DNA repair capacities of healthy and transformed tissue may be exploited to obtain highly selective chemotherapies. We show that the novel N3-(2-fluoroethyl)imidazotetrazine "KL-50" is a selective toxin toward tumors that lack the DNA repair protein O6-methylguanine-DNA-methyltransferase (MGMT), which reverses the formation of O6-alkylguanine lesions. We establish that KL-50 generates DNA interstrand cross-links (ICLs) by a multistep process comprising DNA alkylation to generate an O6-(2-fluoroethyl)guanine (O6FEtG) lesion, slow unimolecular displacement of fluoride to form an N1,O6-ethanoguanine (N1,O6EtG) intermediate, and ring-opening by the adjacent cytidine. The slow rate of N1,O6EtG formation allows healthy cells expressing MGMT to reverse the initial O6FEtG lesion before it evolves to N1,O6EtG, thereby suppressing the formation of toxic DNA-MGMT cross-links and reducing the amount of DNA ICLs generated in healthy cells. In contrast, O6-(2-chloroethyl)guanine lesions produced by agents such as lomustine and the N3-(2-chloroethyl)imidazotetrazine mitozolomide rapidly evolve to N1,O6EtG, resulting in the formation of DNA-MGMT cross-links and DNA ICLs in healthy tissue. These studies suggest that careful consideration of the rates of chemical DNA modification and biochemical DNA repair may lead to the identification of other tumor-specific genotoxic agents.
Assuntos
Neoplasias Encefálicas , Resistencia a Medicamentos Antineoplásicos , Humanos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/síntese química , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Reparo do DNA/efeitos dos fármacos , O(6)-Metilguanina-DNA Metiltransferase/metabolismo , O(6)-Metilguanina-DNA Metiltransferase/antagonistas & inibidores , Imidazóis/química , Imidazóis/farmacologia , Imidazóis/uso terapêuticoRESUMO
The ability to understand and predict variable responses to therapeutic agents may improve outcomes in patients with cancer. We hypothesized that the basal gene-transcription state of cancer cell lines, coupled with cell viability profiles of small molecules, might be leveraged to nominate specific mechanisms of intrinsic resistance and to predict drug combinations that overcome resistance. We analyzed 564,424 sensitivity profiles to identify candidate gene-compound pairs, and validated nine such relationships. We determined the mechanism of a novel relationship, in which expression of the serine hydrolase enzymes monoacylglycerol lipase (MGLL) or carboxylesterase 1 (CES1) confers resistance to the histone lysine demethylase inhibitor GSK-J4 by direct enzymatic modification. Insensitive cell lines could be sensitized to GSK-J4 by inhibition or gene knockout. These analytical and mechanistic studies highlight the potential of integrating gene-expression features with small-molecule response to identify patient populations that are likely to benefit from treatment, to nominate rational candidates for combinations and to provide insights into mechanisms of action.
Assuntos
Histona Desmetilases , Monoacilglicerol Lipases , Biomarcadores , Sobrevivência Celular , Combinação de Medicamentos , Histona Desmetilases/metabolismo , HumanosRESUMO
Species of the genus Blautia are typical inhabitants of the human gut and considered as beneficial gut microbes. However, their role in the gut microbiome and their metabolic features are poorly understood. Blautia schinkii was described as an acetogenic bacterium, characterized by a functional Wood-Ljungdahl pathway (WLP) of acetogenesis from H2 + CO2 . Here we report that two relatives, Blautia luti and Blautia wexlerae do not grow on H2 + CO2 . Inspection of the genome sequence revealed all genes of the WLP except genes encoding a formate dehydrogenase and an electron-bifurcating hydrogenase. Enzyme assays confirmed this prediction. Accordingly, resting cells neither converted H2 + CO2 nor H2 + HCOOH + CO2 to acetate. Carbon monoxide is an intermediate of the WLP and substrate for many acetogens. Blautia luti and B. wexlerae had an active CO dehydrogenase and resting cells performed acetogenesis from HCOOH + CO2 + CO, demonstrating a functional WLP. Bioinformatic analyses revealed that many Blautia strains as well as other gut acetogens lack formate dehydrogenases and hydrogenases. Thus, the use of formate instead of H2 + CO2 as an interspecies hydrogen and electron carrier seems to be more common in the gut microbiome.
Assuntos
Formiato Desidrogenases , Hidrogenase , Proteínas de Bactérias/metabolismo , Dióxido de Carbono/metabolismo , Clostridiales , Formiato Desidrogenases/genética , Humanos , Hidrogenase/genética , Madeira/metabolismoRESUMO
We present a method to rapidly identify hydrogen-mediated interactions in proteins (e.g., hydrogen bonds, hydrogen bonds, water-mediated hydrogen bonds, salt bridges, and aromatic π-hydrogen interactions) through heavy atom geometry alone, that is, without needing to explicitly determine hydrogen atom positions using either experimental or theoretical methods. By including specific real (or virtual) partner atoms as defined by the atom type of both the donor and acceptor heavy atoms, a set of unique angles can be rapidly calculated. By comparing the distance between the donor and the acceptor and these unique angles to the statistical preferences observed in the Protein Data Bank (PDB), we were able to identify a set of conserved geometries (15 for donor atoms and 7 for acceptor atoms) for hydrogen-mediated interactions in proteins. This set of identified interactions includes every polar atom type present in the Protein Data Bank except OE1 (glutamate/glutamine sidechain) and a clear geometric preference for the methionine sulfur atom (SD) to act as a hydrogen bond acceptor. This method could be readily applied to protein design efforts.
Assuntos
Hidrogênio/química , Conformação Molecular , Proteínas/química , Ligação de HidrogênioRESUMO
OBJECTIVES: To describe the use of low-dose bolus epinephrine in critically ill children during an acute hypotensive episode or prearrest condition. DESIGN: Institutional Review Board approved, single-center, retrospective medical chart review. SETTING: Large medical-surgical PICU within a freestanding, tertiary care children's hospital. PATIENTS: Patients admitted to the PICU between June 1, 2015, and June 1, 2016, who received low-dose (≤ 5 µg/kg) IV bolus epinephrine. INTERVENTIONS: None. MEASUREMENT AND MAIN RESULTS: Twenty-four resuscitation episodes (63 doses; 19 patients) were analyzed. Median age and weight of patients were 9 years (interquartile range, 1-15 yr) and 38.5 kg (interquartile range, 12-54.8 kg). Median Pediatric Risk of Mortality III score was 17 (interquartile range, 10-27). Mean epinephrine dose was 1.3 ± 1.1 µg/kg. Median number of doses per patient was two. If more than one dose was provided, median dosing interval was 6.5 minutes. Heart rate and mean arterial blood pressure were compared at the time of epinephrine administration and 1-4 minutes (median = 1 min) following administration. Heart rate changed from 130 ± 41 to 150 ± 33 beats/min (p < 0.05), and mean arterial blood pressure changed from 51 ± 17 to 75 ± 27 mm Hg (p < 0.001). Variability in mean arterial blood pressure response was observed; nonresponders required extracorporeal membrane oxygenation; 66% of doses resulted in up to 100% mean arterial blood pressure increase, and 21% of doses resulted in greater than 100% mean arterial blood pressure increase. Doses below 1 µg/kg were associated with a lower mean arterial blood pressure increase than doses between 1 and 5 µg/kg (mean percent change in mean arterial blood pressure = 6.6% vs 60%, respectively). Children less than or equal to 2 years old had the greatest percentage increase in heart rate and mean arterial blood pressure. CONCLUSIONS: Provision of low-dose bolus epinephrine during periods of acute hypotension can result in a significant increase in mean arterial blood pressure and heart rate. This dosing strategy may provide temporary stabilization while other therapies are added or adjusted, but further research is needed.
Assuntos
Epinefrina/administração & dosagem , Hipotensão/tratamento farmacológico , Vasoconstritores/administração & dosagem , Doença Aguda , Adolescente , Pressão Sanguínea/efeitos dos fármacos , Criança , Pré-Escolar , Epinefrina/efeitos adversos , Feminino , Frequência Cardíaca/efeitos dos fármacos , Humanos , Lactente , Injeções Intravenosas , Unidades de Terapia Intensiva Pediátrica , Masculino , Ressuscitação/métodos , Estudos Retrospectivos , Vasoconstritores/efeitos adversosRESUMO
The histone lysine demethylases KDM4A-C are involved in physiologic processes including stem cell identity and self-renewal during development, DNA damage repair, and cell-cycle progression. KDM4A-C are overexpressed and associated with malignant cell behavior in multiple human cancers and are therefore potential therapeutic targets. Given the role of KDM4A-C in development and cancer, we aimed to test the potent, selective KDM4A-C inhibitor QC6352 on oncogenic cells of renal embryonic lineage. The anaplastic Wilms tumor cell line WiT49 and the tumor-forming human embryonic kidney cell line HEK293 demonstrated low nanomolar QC6352 sensitivity. The cytostatic response to QC6352 in WiT49 and HEK293 cells was marked by induction of DNA damage, a DNA repair-associated protein checkpoint response, S-phase cell-cycle arrest, profound reduction of ribosomal protein gene and rRNA transcription, and blockade of newly synthesized proteins. QC6352 caused reduction of KDM4A-C levels by a proteasome-associated mechanism. The cellular phenotype caused by QC6352 treatment of reduced migration, proliferation, tumor spheroid growth, DNA damage, and S-phase cell-cycle arrest was most closely mirrored by knockdown of KDM4A as determined by siRNA knockdown of KDM4A-C. QC6352 sensitivity correlated with high basal levels of ribosomal gene transcription in more than 900 human cancer cell lines. Targeting KDM4A may be of future therapeutic interest in oncogenic cells of embryonic renal lineage or cells with high basal expression of ribosomal protein genes.
Assuntos
Compostos Heterocíclicos de 4 ou mais Anéis , Histona Desmetilases com o Domínio Jumonji , Proteínas Ribossômicas , Humanos , Células HEK293 , Histona Desmetilases com o Domínio Jumonji/genética , Linhagem Celular Tumoral , Rim/metabolismo , Ribossomos/metabolismoRESUMO
Citron kinase (CITK) is an AGC-family serine/threonine kinase that regulates cytokinesis. Despite knockdown experiments implicating CITK as an anticancer target, no selective CITK inhibitors exist. We transformed a previously reported kinase inhibitor with weak off-target CITK activity into a first-in-class CITK chemical probe, C3TD879. C3TD879 is a Type I kinase inhibitor which potently inhibits CITK catalytic activity (biochemical IC50 = 12 nM), binds directly to full-length human CITK in cells (NanoBRET Kd < 10 nM), and demonstrates favorable DMPK properties for in vivo evaluation. We engineered exquisite selectivity for CITK (>17-fold versus 373 other human kinases), making C3TD879 the first chemical probe suitable for interrogating the complex biology of CITK. Our small-molecule CITK inhibitors could not phenocopy the effects of CITK knockdown in cell proliferation, cell cycle progression, or cytokinesis assays, providing preliminary evidence that the structural roles of CITK may be more important than its kinase activity.
Assuntos
Citocinese , Proteínas Serina-Treonina Quinases , Humanos , Proteínas Serina-Treonina Quinases/metabolismo , Divisão Celular , Citocinese/fisiologia , Fosforilação , Proliferação de CélulasRESUMO
Transcription factors (TFs) are important mediators of aberrant transcriptional programs in cancer cells. In this study, we focus on TF activity (TFa) as a biomarker for cell-line-selective anti-proliferative effects, in that high TFa predicts sensitivity to loss of function of a given gene (i.e., genetic dependencies [GDs]). Our linear-regression-based framework identifies 3,047 pan-cancer and 3,952 cancer-type-specific candidate TFa-GD associations from cell line data, which are then cross-examined for impact on survival in patient cohorts. One of the most prominent biomarkers is TEAD1 activity, whose associations with its predicted GDs are validated through experimental evidence as proof of concept. Overall, these TFa-GD associations represent an attractive resource for identifying innovative, biomarker-driven hypotheses for drug discovery programs in oncology.
Assuntos
Neoplasias , Fatores de Transcrição , Humanos , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Linhagem Celular Tumoral , Fatores de Transcrição de Domínio TEA/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Biomarcadores Tumorais/metabolismo , Biomarcadores Tumorais/genética , Proliferação de CélulasRESUMO
Medulloblastoma is one of the most common malignant brain tumors of children, and 30% of medulloblastomas are driven by gain-of-function genetic lesions in the Sonic Hedgehog (SHH) signaling pathway. EYA1, a haloacid dehalogenase phosphatase and transcription factor, is critical for tumorigenesis and proliferation of SHH medulloblastoma (SHH-MB). Benzarone and benzbromarone have been identified as allosteric inhibitors of EYA proteins. Using benzarone as a point of departure, we developed a panel of 35 derivatives and tested them in SHH-MB. Among these compounds, DS-1-38 functioned as an EYA antagonist and opposed SHH signaling. DS-1-38 inhibited SHH-MB growth in vitro and in vivo, showed excellent brain penetrance, and increased the lifespan of genetically engineered mice predisposed to fatal SHH-MB. These data suggest that EYA inhibitors represent promising therapies for pediatric SHH-MB. SIGNIFICANCE: Development of a benzarone derivative that inhibits EYA1 and impedes the growth of SHH medulloblastoma provides an avenue for improving treatment of this malignant pediatric brain cancer.
Assuntos
Benzobromarona/análogos & derivados , Neoplasias Encefálicas , Neoplasias Cerebelares , Meduloblastoma , Animais , Camundongos , Humanos , Criança , Proteínas Hedgehog , Meduloblastoma/tratamento farmacológico , Meduloblastoma/genética , Neoplasias Cerebelares/tratamento farmacológicoRESUMO
Ubiquitin is a small, highly conserved protein that acts as a posttranslational modification in eukaryotes. Ubiquitination of proteins frequently serves as a degradation signal, marking them for disposal by the proteasome. Here, we report a novel small molecule from a diversity-oriented synthesis library, BRD1732, that is directly ubiquitinated in cells, resulting in dramatic accumulation of inactive ubiquitin monomers and polyubiquitin chains causing broad inhibition of the ubiquitin-proteasome system. Ubiquitination of BRD1732 and its associated cytotoxicity are stereospecific and dependent upon two homologous E3 ubiquitin ligases, RNF19A and RNF19B. Our finding opens the possibility for indirect ubiquitination of a target through a ubiquitinated bifunctional small molecule, and more broadly raises the potential for posttranslational modification in trans.
RESUMO
Cyclin-dependent kinase 9 (CDK9) coordinates signaling events that regulate RNA polymerase II (Pol II) pause-release states. It is an important co-factor for transcription factors, such as MYC, that drive aberrant cell proliferation when their expression is deregulated. CDK9 modulation offers an approach for attenuating dysregulation in such transcriptional programs. As a result, numerous drug development campaigns to inhibit CDK9 kinase activity have been pursued. More recently, targeted degradation has emerged as an attractive approach. However, comprehensive evaluation of degradation versus inhibition is still critically needed to assess the biological contexts in which degradation might offer superior therapeutic benefits. We validated that CDK9 inhibition triggers a compensatory mechanism that dampens its effect on MYC expression and found that this feedback mechanism was absent when the kinase is degraded. Importantly, CDK9 degradation is more effective than its inhibition for disrupting MYC transcriptional regulatory circuitry likely through the abrogation of both enzymatic and scaffolding functions of CDK9. Highlights: - KI-CDK9d-32 is a highly potent and selective CDK9 degrader. - KI-CDK9d-32 leads to rapid downregulation of MYC protein and mRNA transcripts levels. - KI-CDK9d-32 represses canonical MYC pathways and leads to a destabilization of nucleolar homeostasis. - Multidrug resistance ABCB1 gene emerged as the strongest resistance marker for the CDK9 PROTAC degrader.
RESUMO
Chemical discovery efforts commonly target individual protein domains. Many proteins, including the EP300/CBP histone acetyltransferases (HATs), contain several targetable domains. EP300/CBP are critical gene-regulatory targets in cancer, with existing high potency inhibitors of either the catalytic HAT domain or protein-binding bromodomain (BRD). A domain-specific inhibitory approach to multidomain-containing proteins may identify exceptional-responding tumor types, thereby expanding a therapeutic index. Here, we discover that targeting EP300/CBP using the domain-specific inhibitors, A485 (HAT) or CCS1477 (BRD) have different effects in select tumor types. Group 3 medulloblastoma (G3MB) cells are especially sensitive to BRD, compared with HAT inhibition. Structurally, these effects are mediated by the difluorophenyl group in the catalytic core of CCS1477. Mechanistically, bromodomain inhibition causes rapid disruption of genetic dependency networks that are required for G3MB growth. These studies provide a domain-specific structural foundation for drug discovery efforts targeting EP300/CBP and identify a selective role for the EP300/CBP bromodomain in maintaining genetic dependency networks in G3MB.