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1.
J Biol Chem ; 295(11): 3431-3446, 2020 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-32005668

RESUMO

Cytotoxic molecules can kill cancer cells by disrupting critical cellular processes or by inducing novel activities. 6-(4-(Diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one (DNMDP) is a small molecule that kills cancer cells by generation of novel activity. DNMDP induces complex formation between phosphodiesterase 3A (PDE3A) and schlafen family member 12 (SLFN12) and specifically kills cancer cells expressing elevated levels of these two proteins. Here, we examined the characteristics and covariates of the cancer cell response to DNMDP. On average, the sensitivity of human cancer cell lines to DNMDP is correlated with PDE3A expression levels. However, DNMDP could also bind the related protein, PDE3B, and PDE3B supported DNMDP sensitivity in the absence of PDE3A expression. Although inhibition of PDE3A catalytic activity did not account for DNMDP sensitivity, we found that expression of the catalytic domain of PDE3A in cancer cells lacking PDE3A is sufficient to confer sensitivity to DNMDP, and substitutions in the PDE3A active site abolish compound binding. Moreover, a genome-wide CRISPR screen identified the aryl hydrocarbon receptor-interacting protein (AIP), a co-chaperone protein, as required for response to DNMDP. We determined that AIP is also required for PDE3A-SLFN12 complex formation. Our results provide mechanistic insights into how DNMDP induces PDE3A-SLFN12 complex formation, thereby killing cancer cells with high levels of PDE3A and SLFN12 expression.


Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias/patologia , Sequência de Bases , Biomarcadores Tumorais/metabolismo , Sistemas CRISPR-Cas/genética , Domínio Catalítico , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/química , Mutação da Fase de Leitura/genética , Genoma , Heterozigoto , Humanos , Ligação Proteica/efeitos dos fármacos , Piridazinas/farmacologia
2.
Nature ; 506(7488): 371-5, 2014 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-24390348

RESUMO

Cervical cancer is responsible for 10-15% of cancer-related deaths in women worldwide. The aetiological role of infection with high-risk human papilloma viruses (HPVs) in cervical carcinomas is well established. Previous studies have also implicated somatic mutations in PIK3CA, PTEN, TP53, STK11 and KRAS as well as several copy-number alterations in the pathogenesis of cervical carcinomas. Here we report whole-exome sequencing analysis of 115 cervical carcinoma-normal paired samples, transcriptome sequencing of 79 cases and whole-genome sequencing of 14 tumour-normal pairs. Previously unknown somatic mutations in 79 primary squamous cell carcinomas include recurrent E322K substitutions in the MAPK1 gene (8%), inactivating mutations in the HLA-B gene (9%), and mutations in EP300 (16%), FBXW7 (15%), NFE2L2 (4%), TP53 (5%) and ERBB2 (6%). We also observe somatic ELF3 (13%) and CBFB (8%) mutations in 24 adenocarcinomas. Squamous cell carcinomas have higher frequencies of somatic nucleotide substitutions occurring at cytosines preceded by thymines (Tp*C sites) than adenocarcinomas. Gene expression levels at HPV integration sites were statistically significantly higher in tumours with HPV integration compared with expression of the same genes in tumours without viral integration at the same site. These data demonstrate several recurrent genomic alterations in cervical carcinomas that suggest new strategies to combat this disease.


Assuntos
Genoma Humano/genética , Mutação/genética , Neoplasias do Colo do Útero/genética , Adenocarcinoma/genética , Adenocarcinoma/virologia , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/virologia , Estudos de Casos e Controles , Proteínas de Ciclo Celular/genética , Subunidade beta de Fator de Ligação ao Core/genética , Variações do Número de Cópias de DNA/genética , Análise Mutacional de DNA , Proteínas de Ligação a DNA/genética , Proteína p300 Associada a E1A/genética , Exoma/genética , Proteínas F-Box/genética , Proteína 7 com Repetições F-Box-WD , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Genômica , Antígenos HLA-B/genética , Humanos , Proteína Quinase 1 Ativada por Mitógeno/genética , Fator 2 Relacionado a NF-E2/genética , Papillomaviridae/genética , Papillomaviridae/fisiologia , Infecções por Papillomavirus/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-ets , Receptor ErbB-2/genética , Fatores de Transcrição/genética , Transcriptoma/genética , Proteína Supressora de Tumor p53/genética , Ubiquitina-Proteína Ligases/genética , Neoplasias do Colo do Útero/virologia , Integração Viral/genética
4.
Proc Natl Acad Sci U S A ; 109(36): 14476-81, 2012 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-22908275

RESUMO

We assessed somatic alleles of six receptor tyrosine kinase genes mutated in lung adenocarcinoma for oncogenic activity. Five of these genes failed to score in transformation assays; however, novel recurring extracellular domain mutations of the receptor tyrosine kinase gene ERBB2 were potently oncogenic. These ERBB2 extracellular domain mutants were activated by two distinct mechanisms, characterized by elevated C-terminal tail phosphorylation or by covalent dimerization mediated by intermolecular disulfide bond formation. These distinct mechanisms of receptor activation converged upon tyrosine phosphorylation of cellular proteins, impacting cell motility. Survival of Ba/F3 cells transformed to IL-3 independence by the ERBB2 extracellular domain mutants was abrogated by treatment with small-molecule inhibitors of ERBB2, raising the possibility that patients harboring such mutations could benefit from ERBB2-directed therapy.


Assuntos
Adenocarcinoma/enzimologia , Neoplasias Pulmonares/enzimologia , Mutação/genética , Receptor ErbB-2/genética , Adenocarcinoma/genética , Adenocarcinoma de Pulmão , Alelos , Animais , Movimento Celular/fisiologia , Clonagem Molecular , Primers do DNA/genética , Dimerização , Immunoblotting , Neoplasias Pulmonares/genética , Camundongos , Células NIH 3T3 , Fosforilação , Estrutura Terciária de Proteína/genética , Retroviridae , Espectrometria de Massas em Tandem
5.
J Med Chem ; 67(11): 8962-8987, 2024 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-38748070

RESUMO

Dysregulation of histone methyl transferase nuclear receptor-binding SET domain 2 (NSD2) has been implicated in several hematological and solid malignancies. NSD2 is a large multidomain protein that carries histone writing and histone reading functions. To date, identifying inhibitors of the enzymatic activity of NSD2 has proven challenging in terms of potency and SET domain selectivity. Inhibition of the NSD2-PWWP1 domain using small molecules has been considered as an alternative approach to reduce NSD2-unregulated activity. In this article, we present novel computational chemistry approaches, encompassing free energy perturbation coupled to machine learning (FEP/ML) models as well as virtual screening (VS) activities, to identify high-affinity NSD2 PWWP1 binders. Through these activities, we have identified the most potent NSD2-PWWP1 binder reported so far in the literature: compound 34 (pIC50 = 8.2). The compounds identified herein represent useful tools for studying the role of PWWP1 domains for inhibition of human NSD2.


Assuntos
Desenho de Fármacos , Histona-Lisina N-Metiltransferase , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Histona-Lisina N-Metiltransferase/metabolismo , Histona-Lisina N-Metiltransferase/química , Ligantes , Humanos , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Relação Estrutura-Atividade , Aprendizado de Máquina , Modelos Moleculares , Domínios Proteicos
6.
Nat Commun ; 12(1): 4375, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34272366

RESUMO

DNMDP and related compounds, or velcrins, induce complex formation between the phosphodiesterase PDE3A and the SLFN12 protein, leading to a cytotoxic response in cancer cells that express elevated levels of both proteins. The mechanisms by which velcrins induce complex formation, and how the PDE3A-SLFN12 complex causes cancer cell death, are not fully understood. Here, we show that PDE3A and SLFN12 form a heterotetramer stabilized by binding of DNMDP. Interactions between the C-terminal alpha helix of SLFN12 and residues near the active site of PDE3A are required for complex formation, and are further stabilized by interactions between SLFN12 and DNMDP. Moreover, we demonstrate that SLFN12 is an RNase, that PDE3A binding increases SLFN12 RNase activity, and that SLFN12 RNase activity is required for DNMDP response. This new mechanistic understanding will facilitate development of velcrin compounds into new cancer therapies.


Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/química , Peptídeos e Proteínas de Sinalização Intracelular/química , Piridazinas/química , Monofosfato de Adenosina/química , Varredura Diferencial de Calorimetria , Domínio Catalítico , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Microscopia Crioeletrônica , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/genética , Endorribonucleases/química , Células HEK293 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Cinética , Espectrometria de Massas , Complexos Multienzimáticos/ultraestrutura , Mutação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Multimerização Proteica , Piridazinas/farmacologia , Proteínas Recombinantes , Tetra-Hidroisoquinolinas/química
7.
J Clin Invest ; 124(4): 1582-6, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24569458

RESUMO

Targeted cancer therapies often induce "outlier" responses in molecularly defined patient subsets. One patient with advanced-stage lung adenocarcinoma, who was treated with oral sorafenib, demonstrated a near-complete clinical and radiographic remission for 5 years. Whole-genome sequencing and RNA sequencing of primary tumor and normal samples from this patient identified a somatic mutation, ARAF S214C, present in the cancer genome and expressed at high levels. Additional mutations affecting this residue of ARAF and a nearby residue in the related kinase RAF1 were demonstrated across 1% of an independent cohort of lung adenocarcinoma cases. The ARAF mutations were shown to transform immortalized human airway epithelial cells in a sorafenib-sensitive manner. These results suggest that mutant ARAF is an oncogenic driver in lung adenocarcinoma and an indicator of sorafenib response.


Assuntos
Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/genética , Antineoplásicos/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Mutação de Sentido Incorreto , Niacinamida/análogos & derivados , Compostos de Fenilureia/uso terapêutico , Proteínas Proto-Oncogênicas A-raf/genética , Adenocarcinoma/enzimologia , Adenocarcinoma de Pulmão , Idoso , Substituição de Aminoácidos , Transformação Celular Neoplásica/genética , DNA de Neoplasias/genética , Feminino , Humanos , Neoplasias Pulmonares/enzimologia , Terapia de Alvo Molecular , Niacinamida/uso terapêutico , Oncogenes , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas c-raf/genética , Sorafenibe
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